Combination IL-2 immunoconjugate therapy

ABSTRACT

The present invention provides combinations of (a) an immunoconjugate comprising a first antibody engineered to have reduced effector function and an effector moiety, and (b) a second antibody engineered to have increased effector function, for use in treating a disease in an individual in need thereof. Further provided are pharmaceutical compositions comprising the combinations, and methods of using them.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Pat. No. 9,526,797, issuedDec. 27, 2016, which claims benefit under 35 U.S.C. § 119 to EuropeanPatent Application No. 12179473.9 filed on Aug. 7, 2012. The entirecontents of each of the foregoing applications are hereby incorporatedby reference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing submitted viaEFS-Web and hereby incorporated by reference in its entirety. Said ASCIIcopy, created on Aug. 7, 2013, is named P4996US_sequence_listing.txt,and is 170,705 bytes in size.

FIELD OF THE INVENTION

The present invention generally relates to immunotherapy. Moreparticularly, the invention concerns antigen-targeted immunoconjugatesand Fc-engineered antibodies for combined use as immunotherapeuticagents. In addition, the invention relates to pharmaceuticalcompositions comprising combinations of said immunoconjugates andantibodies and methods of using the same in the treatment of disease.

BACKGROUND

The selective destruction of an individual cell or a specific cell typeis often desirable in a variety of clinical settings. For example, it isa primary goal of cancer therapy to specifically destroy tumor cells,while leaving healthy cells and tissues intact and undamaged.

An attractive way of achieving this is by inducing an immune responseagainst the tumor, to make immune effector cells such as natural killer(NK) cells or cytotoxic T lymphocytes (CTLs) attack and destroy tumorcells. Effector cells can be activated by various stimuli, including anumber of cytokines that induce signaling events through binding totheir receptors on the surface of immune cells. For exampleinterleukin-2 (IL-2), which, inter alia, stimulates proliferation andactivation of cytotoxic T cells and NK cells, has been approved for thetreatment of metastatic renal cell carcinoma and malignant melanoma.However, rapid blood clearance and lack of tumor specificity requiresystemic administration of high doses of a cytokine in order to achievea sufficiently high concentration of the cytokine at the tumor site toactivate an immune response or have an anti-tumor effect. These highsystemic levels of cytokine can lead to severe toxicity and adversereactions, as is the case also for IL-2. For use in cancer therapy, itis therefore desirable to specifically deliver cytokines to the tumor ortumor microenvironment. This can be achieved by conjugating the cytokineto a targeting moiety, e.g. an antibody or an antibody fragment,specific for a tumor antigen. A further advantage of suchimmunoconjugates is their increased serum half-life compared to theunconjugated cytokine. Their ability to maximize immunostimulatoryactivities at the site of a tumor whilst keeping systemic side effectsto a minimum at a lower dose makes cytokine immunoconjugates optimalimmunotherapeutic agents.

Another way of activating effector cells is through the engagement ofactivating Fc receptors on their surface by the Fc portion ofimmunoglobulins or recombinant fusion proteins comprising an Fc region.The so-called effector functions of an antibody which are mediated byits Fc region are an important mechanism of action in antibody-basedcancer immunotherapy. Antibody-dependent cell-mediated cytotoxicity, thedestruction of antibody-coated target cells (e.g. tumor cells) by NKcells, is triggered when antibody bound to the surface of a cellinteracts with Fc receptors on the NK cell. NK cells express FcγRIIIa(CD16a) which recognizes immunoglobulins of the IgG₁ or IgG₃ subclass.Further effector functions include antibody-dependent cell-mediatedphagocytosis (ADCP) and complement dependent cytotoxicity (CDC), andvary with the class and subclass of the antibody since different immunecell types bear different sets of Fc receptors which recognize differenttypes and subtypes of immunoglobulin heavy chain constant domains (e.g.α, δ, γ, ε, or μ heavy chain constant domains, corresponding to IgA,IgD, IgE, IgG, or IgM class antibodies, respectively). Variousstrategies have been employed to increase the effector functions ofantibodies. For example, Shields et al. (J Biol Chem 9(2), 6591-6604(2001)) show that amino acid substitutions at positions 298, 333, and/or334 of the Fc region (EU numbering of residues) improve the binding ofantibodies to FcγIIIa receptor and ADCC. Further antibody variantshaving amino acid modifications in the Fc region and exhibiting improvedFc receptor binding and effector function are described e.g. in U.S.Pat. No. 6,737,056, WO 2004/063351 and WO 2004/099249. Alternatively,increased Fc receptor binding and effector function can be obtained byaltering the glycosylation of an antibody. IgGl type antibodies, themost commonly used antibodies in cancer immunotherapy, have a conservedN-linked glycosylation site at Asn 297 in each CH2 domain of the Fcregion. The two complex biantennary oligosaccharides attached to Asn 297are buried between the CH2 domains, forming extensive contacts with thepolypeptide backbone, and their presence is essential for the antibodyto mediate effector functions including antibody-dependent cell-mediatedcytotoxicity (ADCC) (Lifely et al., Glycobiology 5, 813-822 (1995);Jefferis et al., Immunol Rev 163, 59-76 (1998); Wright and Morrison,Trends Biotechnol 15, 26-32 (1997)). Protein engineering studies haveshown that FcγRs interact with the lower hinge region of the IgG CH2domain (Lund et al., J Immunol 157, 4963-69 (1996)). However, FcγRbinding also requires the presence of the oligosaccharides in the CH2region (Lund et al., J Immunol 157, 4963-69 (1996); Wright and Morrison,Trends Biotech 15, 26-31 (1997)), suggesting that either oligosaccharideand polypeptide both directly contribute to the interaction site or thatthe oligosaccharide is required to maintain an active CH2 polypeptideconformation. Modification of the oligosaccharide structure cantherefore be explored as a means to increase the affinity of theinteraction between IgG₁ and FcγR, and to increase ADCC activity of IgG₁antibodies. Umaña et al. (Nat Biotechnol 17, 176-180 (1999) and U.S.Pat. No. 6,602,684 (WO 99/54342), the contents of which are herebyincorporated by reference in their entirety) showed that overexpressionof β(1,4)-N-acetylglucosaminyltransferase III (GnTIII), aglycosyltransferase catalyzing the formation of bisectedoligosaccharides, in Chinese hamster ovary (CHO) cells significantlyincreases the in vitro ADCC activity of antibodies produced in thosecells. Overexpression of GnTIII in production cell lines leads toantibodies enriched in bisected oligosaccharides, which are generallyalso non-fucosylated and of the hybrid type. If in addition to GnTIII,mannosidase II (ManII) is overexpressed in production cell lines,antibodies enriched in bisected, non-fucosylated oligosaccharides of thecomplex type are obtained (Ferrara et al., Biotechn Bioeng 93, 851-861(2006)). Both types of antibodies show strongly increased ADCC, ascompared to antibodies with unmodified glycans, but only antibodies inwhich the majority of the N-glycans are of the complex type are able toinduce significant complement-dependent cytotoxicity (Ferrara et al.,Biotechn Bioeng 93, 851-861 (2006)). The critical factor for theincrease of ADCC activity appears to be the elimination of fucose fromthe innermost N-acetylglucosamine residue of the oligosaccharide core,which improves binding of the IgG Fc domain to FcγRIIIa (Shinkawa etal., J Biol Chem 278, 3466-3473 (2003)). Further methods for producingantibodies with reduced fucosylation include, e.g. expression inα(1,6)-fucosyltransferase deficient host cells (Yamane-Ohnuki et al.,Biotech Bioeng 87, 614-622 (2004); Niwa et al., J Immunol Methods 306,151-160 (2006)).

Despite the successes achieved in anti-cancer immunotherapy by the useof free cytokines, immunoconjugates or engineered antibodies, there is acontinuous need for novel efficacious and safe treatment options incancer therapy.

SUMMARY OF THE INVENTION

The present inventors have found that the combination of these twostrategies for local immune cell activation, i.e. simultaneousstimulation of effector cells by cytokine immunoconjugates and byantibodies engineered to have increased effector functions, greatlyimproves the efficacy of anti-cancer immunotherapy.

Accordingly, the present invention provides a combination of (a) animmunoconjugate comprising a first antibody engineered to have reducedeffector function and an effector moiety, and (b) a second antibodyengineered to have increased effector function, for use in treating adisease in an individual in need thereof. In one embodiment the effectormoiety is a cytokine. In one embodiment the cytokine is selected fromthe group consisting of IL-2, GM-CSF, IFN-α, and IL-12. In a particularembodiment the effector moiety is IL-2. In another embodiment theeffector moiety is IL-12. In another particular embodiment the IL-2effector moiety is a mutant IL-2 effector moiety comprising at least oneamino acid mutation, particularly an amino acid substitution, thatreduces or abolishes the affinity of the mutant IL-2 effector moiety tothe α-subunit of the IL-2 receptor but preserves the affinity of themutant IL-2 effector moiety to the intermediate-affinity IL-2 receptor,compared to the non-mutated IL-2 effector moiety. In a specificembodiment, the mutant IL-2 effector moiety comprises one, two or threeamino acid substitutions at one, two or three position(s) selected fromthe positions corresponding to residue 42, 45, and 72 of human IL-2 (SEQID NO: 1). In a more specific embodiment, the mutant IL-2 effectormoiety comprises three amino acid substitutions at the positionscorresponding to residue 42, 45 and 72 of human IL-2. In an even morespecific embodiment, the mutant IL-2 effector moiety is human IL-2comprising the amino acid substitutions F42A, Y45A and L72G. In certainembodiments the mutant IL-2 effector moiety additionally comprises anamino acid mutation at a position corresponding to position 3 of humanIL-2, which eliminates the O-glycosylation site of IL-2. In a specificembodiment the mutant IL-2 effector moiety comprises the amino acidsequence of SEQ ID NO: 2. In one embodiment the effector moiety is asingle-chain effector moiety.

In one embodiment the first antibody is a full-length IgG classantibody, particularly a full-length IgG₁ sub-class antibody. In oneembodiment the effector moiety shares an amino- or carboxy-terminalpeptide bond with the first antibody. In one embodiment the effectormoiety shares an amino-terminal peptide bond with the first antibody. Inone embodiment, the effector moiety is fused at its N-terminus to theC-terminus of one of the heavy chains of the first antibody. In aparticular embodiment, the immunoconjugate comprises not more than oneeffector moiety. In one embodiment the immunoconjugate essentiallyconsists of an effector moiety and a first antibody joined by one ormore peptide linkers. In a specific embodiment the immunoconjugatecomprises an effector moiety, particularly a single chain effectormoiety, and a first antibody, particularly a full-length IgG classantibody, wherein the effector moiety is fused at its amino-terminalamino acid to the carboxy-terminus of one of the heavy chains of thefirst antibody, optionally through a peptide linker. In certainembodiments the first antibody comprises in the Fc region a modificationpromoting heterodimerization of two non-identical immunoglobulin heavychains. In a specific embodiment said modification is a knob-into-holemodification, comprising a knob modification in one of theimmunoglobulin heavy chains and a hole modification in the other one ofthe two immunoglobulin heavy chains. In one embodiment, the firstantibody comprises a modification within the interface between the twoimmunoglobulin heavy chains in the CH3 domain, wherein i) in the CH3domain of one heavy chain, an amino acid residue is replaced with anamino acid residue having a larger side chain volume, thereby generatinga protuberance (“knob”) within the interface in the CH3 domain of oneheavy chain which is positionable in a cavity (“hole”) within theinterface in the CH3 domain of the other heavy chain, and ii) in the CH3domain of the other heavy chain, an amino acid residue is replaced withan amino acid residue having a smaller side chain volume, therebygenerating a cavity (“hole”) within the interface in the second CH3domain within which a protuberance (“knob”) within the interface in thefirst CH3 domain is positionable. In one embodiment, the first antibodycomprises the amino acid substitution T366W and optionally the aminoacid substitution S354C in one of the immunoglobulin heavy chains, andthe amino acid substitutions T366S, L368A, Y407V and optionally Y349C inthe other one of the immunoglobulin heavy chains. In a particularembodiment the effector moiety is fused to the amino- orcarboxy-terminal amino acid of the immunoglobulin heavy chain comprisingthe knob modification.

In one embodiment the reduced effector function of the first antibody isselected from the group of reduced binding to an activating Fc receptor,reduced ADCC, reduced ADCP, reduced CDC, and reduced cytokine secretion.In one embodiment the reduced effector function is reduced binding to anactivating Fc receptor. In one embodiment the activating Fc receptor isa human receptor. In one embodiment the activating Fc receptor is an Fcγreceptor. In a specific embodiment the activating Fc receptor isselected from the group of FcγRIIIa, FcγRI, and FcRγIIa. In oneembodiment the activating Fc receptor is FcγRIIIa, particularly humanFcγRIIIa. In one embodiment the reduced effector function is reducedADCC. In one embodiment the reduced effector function is reduced bindingto an activating Fc receptor and reduced ADCC.

In one embodiment the first antibody is engineered by introduction ofone or more amino acid mutations in the Fc region. In a specificembodiment the amino acid mutations are amino acid substitutions. In aspecific embodiment, the first antibody, particularly a humanfull-length IgG₁ sub-class antibody, comprises an amino acidsubstitution at position P329 of the immunoglobulin heavy chains (Kabatnumbering). In a more specific embodiment the amino acid substitution isP329A or P329G, particularly P329G. In one embodiment the antibodycomprises a further amino acid substitution at a position selected fromS228, E233, L234, L235, N297 and P331 of the immunoglobulin heavychains. In a more specific embodiment the further amino acidsubstitution is S228P, E233P, L234A, L235A, L235E, N297A, N297D orP331S. In a particular embodiment the antibody comprises amino acidsubstitutions at positions P329, L234 and L235 of the immunoglobulinheavy chains (Kabat numbering). In a more particular embodiment theantibody comprises the amino acid substitutions L234A, L235A and P329G(LALA P329G) in the immunoglobulin heavy chains.

In certain embodiments the first antibody is directed to an antigenpresented on a tumor cell or in a tumor cell environment. In a specificembodiment the first antibody is directed to an antigen selected fromthe group of Fibroblast Activation Protein (FAP), the A1 domain ofTenascin-C (TNC A1), the A2 domain of Tenascin-C (TNC A2), the ExtraDomain B of Fibronectin (EDB), Carcinoembryonic Antigen (CEA) andMelanoma-associated Chondroitin Sulfate Proteoglycan (MCSP). In aparticular embodiment the first antibody is directed to CEA. In anotherparticular embodiment, the first antibody is directed to FAP.

In one embodiment the increased effector function of the second antibodyis selected from the group of increased binding to an activating Fcreceptor, increased ADCC, increased ADCP, increased CDC, and increasedcytokine secretion. In one embodiment the increased effector function isincreased binding to an activating Fc receptor. In a specific embodimentthe activating Fc receptor is selected from the group of FcγRIIIa,FcγRI, and FcRγIIa. In one embodiment the activating Fc receptor isFcγRIIIa. In one embodiment the increased effector function is increasedADCC. In one embodiment the increased effector function is increasedbinding to an activating Fc receptor and increased ADCC.

In one embodiment the second antibody is engineered by introduction ofone or more amino acid mutations in the Fc region. In a specificembodiment the amino acid mutations are amino acid substitutions. In oneembodiment the second antibody is engineered by modification of theglycosylation in the Fc region. In a specific embodiment themodification of the glycosylation in the Fc region is an increasedproportion of non-fucosylated oligosaccharides in the Fc region, ascompared to a non-engineered antibody. In an even more specificembodiment the increased proportion of non-fucosylated oligosaccharidesin the Fc region is at least 20%, preferably at least 50%, mostpreferably at least 70% of non-fucosylated oligosaccharides in the Fcregion. In another specific embodiment the modification of theglycosylation in the Fc region is an increased proportion of bisectedoligosaccharides in the Fc region, as compared to a non-engineeredantibody. In an even more specific embodiment the increased proportionof bisected oligosaccharides in the Fc region is at least about 20%,preferably at least 50%, and most preferably at least 70% of bisectedoligosaccharides in the Fc region. In yet another specific embodimentthe modification of the glycosylation in the Fc region is an increasedproportion of bisected, non-fucosylated oligosaccharides in the Fcregion, as compared to a non-engineered antibody. Preferably the secondantibody has at least about 25%, at least about 35%, or at least about50% of bisected, non-fucosylated oligosaccharides in the Fc region. In aparticular embodiment the second antibody is engineered to have anincreased proportion of non-fucosylated oligosaccharides in the Fcregion as compared to a non-engineered antibody. An increased proportionof non-fucosylated oligosaccharides in the Fc region of an antibodyresults in the antibody having increased effector function, inparticular increased ADCC. In a particular embodiment thenon-fucosylated oligosaccharides are bisected, non-fucosylatedoligosaccharides.

In one embodiment the second antibody is a full-length IgG classantibody, particularly a full-length IgG₁ subclass antibody. In certainembodiments the second antibody is directed to an antigen presented on atumor cell. In a specific embodiment the second antibody is directed toan antigen selected from the group of CD20, Epidermal Growth FactorReceptor (EGFR), HER2, HER3, Insulin-like Growth Factor 1 Receptor(IGF-1R), c-Met, CUB domain-containing protein-1 (CDCP1),Carcinoembryonic Antigen (CEA) and Melanoma-associated ChondroitinSulfate Proteoglycan (MCSP).

In a particular embodiment the second antibody is an anti-CD20 antibodyengineered to have an increased proportion of non-fucosylatedoligosaccharides in the Fc region as compared to a non-engineeredantibody. Suitable anti-CD20 antibodies are described in WO 2005/044859,which is incorporated herein by reference in its entirety. In anotherparticular embodiment the second antibody is an anti-EGFR antibodyengineered to have an increased proportion of non-fucosylatedoligosaccharides in the Fc region as compared to a non-engineeredantibody. Suitable anti-EGFR antibodies are described in WO 2006/082515and WO 2008/017963, each of which is incorporated herein by reference inits entirety. In a further particular embodiment the second antibody isan anti-IGF-1R antibody engineered to have an increased proportion ofnon-fucosylated oligosaccharides in the Fc region as compared to anon-engineered antibody. Suitable anti-IGF-1R antibodies are describedin WO 2008/077546, which is incorporated herein by reference in itsentirety. In yet another particular embodiment the second antibody is ananti-CEA antibody engineered to have an increased proportion ofnon-fucosylated oligosaccharides in the Fc region as compared to anon-engineered antibody. Suitable anti-CEA antibodies are described inPCT publication number WO 2011/023787, which is incorporated herein byreference in its entirety. In yet another particular embodiment thesecond antibody is an anti-HER3 antibody engineered to have an increasedproportion of non-fucosylated oligosaccharides in the Fc region ascompared to a non-engineered antibody. Suitable anti-HER3 antibodies aredescribed in PCT publication number WO 2011/076683, which isincorporated herein by reference in its entirety. In yet anotherparticular embodiment the second antibody is an anti-CDCP1 antibodyengineered to have an increased proportion of non-fucosylatedoligosaccharides in the Fc region as compared to a non-engineeredantibody. Suitable anti-CDCP1 antibodies are described in PCTpublication number WO 2011/023389, which is incorporated herein byreference in its entirety. In one embodiment the second antibody isengineered to have modified glycosylation in the Fc region, as comparedto a non-engineered antibody, by producing the antibody in a host cellhaving altered activity of one or more glycosyltransferase.

In one embodiment the second antibody is engineered to have an increasedproportion of non-fucosylated oligosaccharides in the Fc region, ascompared to a non-engineered antibody, by producing the antibody in ahost cell having increased β(1,4)-N-acetylglucosaminyltransferase III(GnTIII) activity. In a particular embodiment the host cell additionallyhas increased α-mannosidase II (ManII) activity. In another embodimentthe second antibody is engineered to have an increased proportion ofnon-fucosylated oligosaccharides in the Fc region, as compared to anon-engineered antibody, by producing the antibody in a host cell havingdecreased α(1,6)-fucosyltransferase activity.

In one embodiment the disease is a disorder treatable by stimulation ofeffector cell function. In one embodiment the disease is a cellproliferation disorder. In a particular embodiment the disease iscancer. In a specific embodiment the cancer is selected from the groupof lung cancer, colorectal cancer, renal cancer, prostate cancer, breastcancer, head and neck cancer, ovarian cancer, brain cancer, lymphoma,leukemia, and skin cancer. In one embodiment the individual is a mammal.In a particular embodiment the individual is a human.

In a particular embodiment, the invention provides a combination of

(a) an immunoconjugate comprising a first full-length IgG class antibodyengineered to have reduced effector function by introduction of one ormore amino acid mutation in the Fc region and a cytokine, wherein theeffector moiety is fused at its amino-terminal amino acid to thecarboxy-terminus of one of the heavy chains of the first antibody,optionally through a peptide linker, and(b) a second full-length IgG class antibody engineered to have increasedeffector function by modification of the glycosylation in the Fc region,for use in treating a disease in an individual in need thereof. Inanother aspect the invention provides a pharmaceutical compositioncomprising (a) an immunoconjugate comprising a first antibody engineeredto have reduced effector function and an effector moiety, and (b) asecond antibody engineered to have increased effector function, in apharmaceutically acceptable carrier.

The invention also encompasses the use of (a) an immunoconjugatecomprising a first antibody engineered to have reduced effector functionand an effector moiety, and (b) a second antibody engineered to haveincreased effector function, for the manufacture of a medicament for thetreatment of a disease in an individual.

The invention further provides a method of treating a disease in anindividual, comprising administering to the individual a combination of(a) an immunoconjugate comprising a first antibody engineered to havereduced effector function and an effector moiety, and (b) a secondantibody engineered to have increased effector function, in atherapeutically effective amount.

Also provided by the invention is a method of stimulating effector cellfunction in an individual, comprising administering to the individual acombination of (a) an immunoconjugate comprising a first antibodyengineered to have reduced effector function and an effector moiety, and(b) a second antibody engineered to have increased effector function, inan amount effective to stimulate effector cell function.

In a further aspect the invention provides a kit intended for thetreatment of a disease, comprising in the same or in separate containers(a) an immunoconjugate comprising a first antibody engineered to havereduced effector function and an effector moiety, (b) a second antibodyengineered to have increased effector function, and (c) optionally apackage insert comprising printed instructions directing the use of thecombined treatment as a method for treating the disease.

It is understood that the immunoconjugate and the second antibody usedin the pharmaceutical composition, use, methods and kit according to theinvention may incorporate any of the features, singly or in combination,described in the preceding paragraphs in relation to the secondantibodies and immunoconjugates useful for the invention.

SHORT DESCRIPTION OF THE DRAWINGS

FIG. 1A-B. The FAP-targeted 28H1 IgG-IL-2 immunoconjugate (A) or theuntargeted DP47GS IgG-IL-2 immunoconjugate (B), comprising the IL-2quadruple mutant (qm) that lacks binding to CD25, and the anti-EGFRGLYCOMAB were tested in the human head and neck carcinoma cell lineFaDu, intralingually injected into SCID mice. The data show that thecombination of the 28H1 IgG-IL2 qm immunoconjugate, but not the DP47GSIgG-IL2 qm immunoconjugate, and the anti-EGFR GLYCOMAB mediates superiorefficacy in terms of enhanced median survival compared to the respectiveimmunoconjugate or the anti-EGFR GLYCOMAB alone (see Example 1).

FIG. 2A-B. Overall A549 tumor cell killing by PBMCs (E:T=10:1, 4 hours),pre-treated or not with 0.57 nM (A) or 5.7 nM (B) FAP-targeted 28H1IgG-IL2 qm immunoconjugate or IL-2 (Proleukin), in the presence ofdifferent concentrations of anti-EGFR GLYCOMAB (see Example 2).

FIG. 3A-B. The CEA-targeted CH1A1A IgG-IL-2 immunoconjugate comprisingthe IL-2 quadruple mutant (qm) that lacks binding to CD25, and theanti-EGFR GLYCOMAB (A) or cetuximab (B) were tested in the humancolorectal carcinoma cell line LS174T, intrasplenically injected intoSCID FcγRIII transgenic mice. The data show that the combination of theCH1A1A IgG-IL2 qm immunoconjugate and the anti-EGFR GLYCOMAB mediatessuperior efficacy in terms of enhanced median and overall survivalcompared to the respective immunoconjugate, the anti-EGFR GLYCOMAB orcetuximab alone, as well as the combination of the CH1A1A IgG-IL2 qmimmunoconjugate and cetuximab (see Example 3).

FIG. 4A-B. The CEA-targeted CH1A1A IgG-IL-2 immunoconjugate comprisingthe IL-2 quadruple mutant (qm) that lacks binding to CD25, and theanti-EGFR GLYCOMAB (A) or cetuximab (B) were tested in the human lungcarcinoma cell line A549, intravenously injected into SCID FcγRIIItransgenic mice. The data show that the combination of the CH1A1AIgG-IL2 qm immunoconjugate and the anti-EGFR GLYCOMAB mediates superiorefficacy in terms of enhanced median and overall survival compared tothe respective immunoconjugate or the anti-EGFR GLYCOMAB alone, as wellas the combination of the CH1A1A IgG-IL2 qm immunoconjugate andcetuximab (see Example 4).

FIG. 5. The CEA-targeted CH1A1A IgG-IL-2 immunoconjugate comprising theIL-2 quadruple mutant (qm) that lacks binding to CD25, and the anti-Her3GLYCOMAB were tested in the human colorectal carcinoma cell line LS174T,intrasplenically injected into SCID FcγRIII transgenic mice. The datashow that the combination of the CH1A1A IgG-IL2 qm immunoconjugate andthe anti-Her3 GLYCOMAB mediates superior efficacy in terms of enhancedmedian survival compared to the respective immunoconjugate or theanti-Her3 GLYCOMAB alone (see Example 5).

FIG. 6. The FAP-targeted 28H1 IgG-IL-2 immunoconjugate comprising theIL-2 quadruple mutant (qm) that lacks binding to CD25, and the anti-EGFRGLYCOMAB were tested in the human renal carcinoma cell line ACHN,intrarenally injected into SCID FcγRIII transgenic mice. The data showthat the combination of the CH1A1A IgG-IL2 qm immunoconjugate and theanti-EGFR GLYCOMAB mediates superior efficacy in terms of enhancedmedian and overall survival compared to the anti-EGFR GLYCOMAB alone, orthe anti-EGFR GLYCOMAB in combination with Proleukin® (see Example 6).

FIG. 7. Overall LS174T cell killing by PBMCs upon treatment withanti-Her3 GLYCOMAB alone (left panel), the CH1A1A IgG-IL-2 qmimmunoconjugate alone (right panel) or the combination of the CH1A1AIgG-IL-2 qm immunoconjugate with the anti-Her3 GLYCOMAB (right panel).

FIG. 8A-B. Expression of CD25 (A) or CD69 (B) on NK cells upon treatmentwith anti-Her3 GLYCOMAB alone (left panel), the CH1A1A IgG-IL-2 qmimmunoconjugate alone (right panel) or the combination of the CH1A1AIgG-IL-2 qm immunoconjugate with the anti-Her3 GLYCOMAB (right panel).

DETAILED DESCRIPTION OF THE INVENTION

In a first aspect the present invention provides a combination of (a) animmunoconjugate comprising a first antibody engineered to have reducedeffector function and an effector moiety, and (b) a second antibodyengineered to have increased effector function, for use in treating adisease in an individual in need thereof.

The invention further provides a method of treating a disease in anindividual, comprising administering to the individual a combination of(a) an immunoconjugate comprising a first antibody engineered to havereduced effector function and an effector moiety, and (b) a secondantibody engineered to have increased effector function, in atherapeutically effective amount.

Also provided by the invention is a method of stimulating effector cellfunction in an individual, comprising administering to the individual acombination of (a) an immunoconjugate comprising a first antibodyengineered to have reduced effector function and an effector moiety, and(b) a second antibody engineered to have increased effector function, inan amount effective to stimulate effector cell function.

Definitions

Terms are used herein as generally used in the art, unless otherwisedefined in the following.

As used herein, the term “immunoconjugate” refers to a polypeptidemolecule that includes at least one effector moiety and an antibody. Incertain embodiments, the immunoconjugate comprises not more than oneeffector moiety. Particular immunoconjugates according to the inventionessentially consist of one effector moiety and an antibody joined by oneor more peptide linkers. Particular immunoconjugates according to theinvention are fusion proteins, i.e. the components of the immunconjugateare joined by peptide bonds.

As used herein, the term “control antibody” refers to an antibody as itwould exist free of effector moieties. For example, when comparing aIgG-IL2 immunoconjugate as described herein with a control antibody, thecontrol antibody is free IgG, wherein the IgG-IL2 immunoconjugate andthe free IgG molecule can both specifically bind to the same antigenicdeterminant.

As used herein, the term “antigenic determinant” is synonymous with“antigen” and “epitope,” and refers to a site (e.g. a contiguous stretchof amino acids or a conformational configuration made up of differentregions of non-contiguous amino acids) on a polypeptide macromolecule towhich an antibody binds, forming an antibody-antigen complex. Usefulantigenic determinants can be found, for example, on the surfaces oftumor cells, on the surfaces of virus-infected cells, on the surfaces ofother diseased cells, free in blood serum, and/or in the extracellularmatrix (ECM).

By “specifically binds” is meant that the binding is selective for theantigen and can be discriminated from unwanted or non-specificinteractions. The ability of an antibody to bind to a specific antigenicdeterminant can be measured either through an enzyme-linkedimmunosorbent assay (ELISA) or other techniques familiar to one of skillin the art, e.g. surface plasmon resonance technique (analyzed on aBIAcore instrument) (Liljeblad et al., Glyco J 17, 323-329 (2000)), andtraditional binding assays (Heeley, Endocr Res 28, 217-229 (2002)).

The terms “anti-[antigen] antibody” and “an antibody that binds to[antigen]” refer to an antibody that is capable of binding therespective antigen with sufficient affinity such that the antibody isuseful as a diagnostic and/or therapeutic agent in targeting theantigen. In one embodiment, the extent of binding of an anti-[antigen]antibody to an unrelated protein is less than about 10% of the bindingof the antibody to the antigen as measured, e.g., by a radioimmunoassay(RIA). In certain embodiments, an antibody that binds to [antigen] has adissociation constant (K_(D)) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM,≤0.01 nM, or ≤0.001 nM (e.g. 10⁻⁸M or less, e.g. from 10⁻⁸M to 10⁻¹³M,e.g., from 10⁻⁹M to 10⁻¹³ M).

“Affinity” refers to the strength of the sum total of non-covalentinteractions between a single binding site of a molecule (e.g., anantibody) and its binding partner (e.g., an antigen). Unless indicatedotherwise, as used herein, “binding affinity” refers to intrinsicbinding affinity which reflects a 1:1 interaction between members of abinding pair (e.g., antibody and antigen). The affinity of a molecule Xfor its partner Y can generally be represented by the dissociationconstant (K_(D)), which is the ratio of dissociation and associationrate constants (k_(off) and k_(on), respectively). Thus, equivalentaffinities may comprise different rate constants, as long as the ratioof the rate constants remains the same. Affinity can be measured by wellestablished methods known in the art, including those described herein.A particular method for measuring affinity is Surface Plasmon Resonance(SPR).

According to one embodiment, K_(D) is measured by surface plasmonresonance using a BIACORE® T100 machine (GE Healthcare) at 25° C. withligand (e.g. effector moiety receptor, Fc receptor or target antigen)immobilized on CM5 chips. Briefly, carboxymethylated dextran biosensorchips (CM5, GE Healthcare) are activated withN-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) andN-hydroxysuccinimide (NHS) according to the supplier's instructions.Recombinant ligand is diluted with 10 mM sodium acetate, pH 5.5, to0.5-30 μg/ml before injection at a flow rate of 10 μl/minute to achieveapproximately 100-5000 response units (RU) of coupled protein. Followingthe injection of the ligand, 1 M ethanolamine is injected to blockunreacted groups. For kinetics measurements, three- to five-fold serialdilutions of immunoconjugate (range between ˜0.01 nM to 300 nM) areinjected in HBS-EP+(GE Healthcare, 10 mM HEPES, 150 mM NaCl, 3 mM EDTA,0.05% Surfactant P20, pH 7.4) at 25° C. at a flow rate of approximately30-50 μl/min. Association rates (k_(on)) and dissociation rates(k_(off)) are calculated using a simple one-to-one Langmuir bindingmodel (BIACORE® T100 Evaluation Software version 1.1.1) bysimultaneously fitting the association and dissociation sensorgrams. Theequilibrium dissociation constant (K_(D)) is calculated as the ratiok_(off)/k_(on). See, e.g., Chen et al., J Mol Biol 293, 865-881 (1999).

“Reduced binding”, for example reduced binding to an Fc receptor or toCD25, refers to a decrease in affinity for the respective interaction,as measured for example by SPR. For clarity the term includes alsoreduction of the affinity to zero (or below the detection limit of theanalytic method), i.e. complete abolishment of the interaction.Conversely, “increased binding” refers to an increase in bindingaffinity for the respective interaction.

As used herein, the terms “first” and “second” with respect toantibodies, effector moieties etc., are used for convenience ofdistinguishing when there is more than one of each type of moiety. Useof these terms is not intended to confer a specific order or orientationof the immunoconjugate unless explicitly so stated.

As used herein, the term “effector moiety” refers to a polypeptide,e.g., a protein or glycoprotein, that influences cellular activity, forexample, through signal transduction or other cellular pathways.Accordingly, the effector moiety of the invention can be associated withreceptor-mediated signaling that transmits a signal from outside thecell membrane to modulate a response in a cell bearing one or morereceptors for the effector moiety. In one embodiment, an effector moietycan elicit a cytotoxic response in cells bearing one or more receptorsfor the effector moiety. In another embodiment, an effector moiety canelicit a proliferative response in cells bearing one or more receptorsfor the effector moiety. In another embodiment, an effector moiety canelicit differentiation in cells bearing receptors for the effectormoiety. In another embodiment, an effector moiety can alter expression(i.e. upregulate or downregulate) of an endogenous cellular protein incells bearing receptors for the effector moiety. Non-limiting examplesof effector moieties include cytokines, growth factors, hormones,enzymes, substrates, and cofactors. The effector moiety can beassociated with an antibody in a variety of configurations to form animmunoconjugate.

As used herein, the term “cytokine” refers to a molecule that mediatesand/or regulates a biological or cellular function or process (e.g.immunity, inflammation, and hematopoiesis). The term “cytokine” as usedherein includes “lymphokines,” “chemokines,” “monokines,” and“interleukins”. Examples of useful cytokines include, but are notlimited to, GM-CSF, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,IL-8, IL-10, IL-12, IFN-α, IFN-β, IFN-γ, MIP-1α, MIP-1β, TGF-β, TNF-α,and TNF-β. Particular cytokines are IL-2 and IL-12. The term “cytokine”as used herein is meant to also include cytokine variants comprising oneor more amino acid mutations in the amino acid sequences of thecorresponding wild-type cytokine, such as for example the IL-2 variantsdescribed in Sauvé et al., Proc Natl Acad Sci USA 88, 4636-40 (1991); Huet al., Blood 101, 4853-4861 (2003) and US Pat. Publ. No. 2003/0124678;Shanafelt et al., Nature Biotechnol 18, 1197-1202 (2000); Heaton et al.,Cancer Res 53, 2597-602 (1993) and U.S. Pat. No. 5,229,109; US Pat.Publ. No. 2007/0036752; WO 2008/0034473; WO 2009/061853; or hereinaboveand -below.

As used herein, the term “single-chain” refers to a molecule comprisingamino acid monomers linearly linked by peptide bonds. In one embodiment,the effector moiety is a single-chain effector moiety. Non-limitingexamples of single-chain effector moieties include cytokines, growthfactors, hormones, enzymes, substrates, and cofactors. When the effectormoiety is a cytokine and the cytokine of interest is normally found as amultimer in nature, each subunit of the multimeric cytokine issequentially encoded by the single-chain of the effector moiety.Accordingly, non-limiting examples of useful single-chain effectormoieties include GM-CSF, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-10, IL-12, IFN-α, IFN-β, IFN-γ, MIP-1α, MIP-1β, TGF-β,TNF-α, and TNF-β.

As used herein, the term “control effector moiety” refers to anunconjugated effector moiety. For example, when comparing an IL-2immunoconjugate as described herein with a control effector moiety, thecontrol effector moiety is free, unconjugated IL-2. Likewise, e.g., whencomparing an IL-12 immunoconjugate with a control effector moiety, thecontrol effector moiety is free, unconjugated IL-12 (e.g. existing as aheterodimeric protein wherein the p40 and p35 subunits share onlydisulfide bond(s)).

As used herein, the term “effector moiety receptor” refers to apolypeptide molecule capable of binding specifically to an effectormoiety. For example, where IL-2 is the effector moiety, the effectormoiety receptor that binds to an IL-2 molecule (e.g. an immunoconjugatecomprising IL-2) is the IL-2 receptor. Similarly, e.g., where IL-12 isthe effector moiety of an immunoconjugate, the effector moiety receptoris the IL-12 receptor. Where an effector moiety specifically binds tomore than one receptor, all receptors that specifically bind to theeffector moiety are “effector moiety receptors” for that effectormoiety.

The term “antibody” herein is used in the broadest sense and encompassesvarious antibody structures, including but not limited to monoclonalantibodies, polyclonal antibodies, multispecific antibodies (e.g.bispecific antibodies), and antibody fragments so long as they exhibitthe desired antigen-binding activity and comprise an Fc region or aregion equivalent to the Fc region of an immunoglobulin.

The terms “full-length antibody,” “intact antibody,” and “wholeantibody” are used herein interchangeably to refer to an antibody havinga structure substantially similar to a native immunoglobulin structure.

The term “immunoglobulin” refers to a protein having the structure of anaturally occurring antibody. For example, immunoglobulins of the IgGclass are heterotetrameric glycoproteins of about 150,000 daltons,composed of two light chains and two heavy chains that aredisulfide-bonded. From N- to C-terminus, each heavy chain has a variableregion (VH), also called a variable heavy domain or a heavy chainvariable domain, followed by three constant domains (CH1, CH2, and CH3),also called a heavy chain constant region. Similarly, from N- toC-terminus, each light chain has a variable region (VL), also called avariable light domain or a light chain variable domain, followed by aconstant light (CL) domain, also called a light chain constant region.The heavy chain of an immunoglobulin may be assigned to one of fivetypes, called α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), some ofwhich may be further divided into subtypes, e.g. γ₁ (IgG₁), γ₂ (IgG₂),γ₃ (IgG₃), γ₄ (IgG₄), α₁ (IgA₁) and α₂ (IgA₂). The light chain of animmunoglobulin may be assigned to one of two types, called kappa (κ) andlambda (λ), based on the amino acid sequence of its constant domain. Animmunoglobulin essentially consists of two Fab molecules and an Fcregion, linked via the immunoglobulin hinge region.

An “antibody fragment” refers to a molecule other than an intactantibody that comprises a portion of an intact antibody that binds theantigen to which the intact antibody binds. Examples of antibodyfragments include but are not limited to Fv, Fab, Fab′, Fab′-SH,F(ab′)₂, diabodies, linear antibodies, single-chain antibody molecules(e.g. scFv), single-domain antibodies, and multispecific antibodiesformed from antibody fragments. For a review of certain antibodyfragments, see Hudson et al., Nat Med 9, 129-134 (2003). For a review ofscFv fragments, see e.g. Plückthun, in The Pharmacology of MonoclonalAntibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, NewYork, pp. 269-315 (1994); see also WO 93/16185; and U.S. Pat. Nos.5,571,894 and 5,587,458. For discussion of Fab and F(ab′)₂ fragmentscomprising salvage receptor binding epitope residues and havingincreased in vivo half-life, see U.S. Pat. No. 5,869,046. Diabodies areantibody fragments with two antigen-binding sites that may be bivalentor bispecific. See, for example, EP 404,097; WO 1993/01161; Hudson etal., Nat Med 9, 129-134 (2003); and Hollinger et al., Proc Natl Acad SciUSA 90, 6444-6448 (1993). Triabodies and tetrabodies are also describedin Hudson et al., Nat Med 9, 129-134 (2003). Single-domain antibodiesare antibody fragments comprising all or a portion of the heavy chainvariable domain or all or a portion of the light chain variable domainof an antibody. In certain embodiments, a single-domain antibody is ahuman single-domain antibody (Domantis, Inc., Waltham, Mass.; see e.g.U.S. Pat. No. 6,248,516 B1). Antibody fragments can be made by varioustechniques, including but not limited to proteolytic digestion of anintact antibody as well as production by recombinant host cells (e.g. E.coli or phage), as described herein.

The term “antigen binding domain” refers to the part of an antibody thatcomprises the area which specifically binds to and is complementary topart or all of an antigen. An antigen binding domain may be provided by,for example, one or more antibody variable domains (also called antibodyvariable regions). Particularly, an antigen binding domain comprises anantibody light chain variable region (VL) and an antibody heavy chainvariable region (VH).

The term “variable region” or “variable domain” refers to the domain ofan antibody heavy or light chain that is involved in binding theantibody to antigen. The variable domains of the heavy chain and lightchain (VH and VL, respectively) of a native antibody generally havesimilar structures, with each domain comprising four conserved frameworkregions (FRs) and three hypervariable regions (HVRs). See, e.g., Kindtet al., Kuby Immunology, 6^(th) ed., W.H. Freeman and Co., page 91(2007). A single VH or VL domain may be sufficient to conferantigen-binding specificity.

The term “hypervariable region” or “HVR”, as used herein, refers to eachof the regions of an antibody variable domain which are hypervariable insequence and/or form structurally defined loops (“hypervariable loops”).Generally, native four-chain antibodies comprise six HVRs; three in theVH (H1, H2, H3), and three in the VL (L1, L2, L3). HVRs generallycomprise amino acid residues from the hypervariable loops and/or fromthe complementarity determining regions (CDRs), the latter being ofhighest sequence variability and/or involved in antigen recognition.With the exception of CDR1 in VH, CDRs generally comprise the amino acidresidues that form the hypervariable loops. Hypervariable regions (HVRs)are also referred to as “complementarity determining regions” (CDRs),and these terms are used herein interchangeably in reference to portionsof the variable region that form the antigen binding regions. Thisparticular region has been described by Kabat et al., U.S. Dept. ofHealth and Human Services, Sequences of Proteins of ImmunologicalInterest (1983) and by Chothia et al., J Mol Biol 196:901-917 (1987),where the definitions include overlapping or subsets of amino acidresidues when compared against each other. Nevertheless, application ofeither definition to refer to a CDR of an antibody or variants thereofis intended to be within the scope of the term as defined and usedherein. The appropriate amino acid residues which encompass the CDRs asdefined by each of the above cited references are set forth below inTable 1 as a comparison. The exact residue numbers which encompass aparticular CDR will vary depending on the sequence and size of the CDR.Those skilled in the art can routinely determine which residues comprisea particular CDR given the variable region amino acid sequence of theantibody.

TABLE 1 CDR Definitions¹ CDR Kabat Chothia AbM² V_(H) CDR1 31-35 26-3226-35 V_(H) CDR2 50-65 52-58 50-58 V_(H) CDR3 95-102 95-102 95-102 V_(L)CDR1 24-34 26-32 24-34 V_(L) CDR2 50-56 50-52 50-56 V_(L) CDR3 89-9791-96 89-97 ¹Numbering of all CDR definitions in Table 1 is according tothe numbering conventions set forth by Kabat et al. (see below). ²“AbM”with a lowercase “b” as used in Table 1 refers to the CDRs as defined byOxford Molecular's “AbM” antibody modeling software.

Kabat et al. also defined a numbering system for variable regionsequences that is applicable to any antibody. One of ordinary skill inthe art can unambiguously assign this system of “Kabat numbering” to anyvariable region sequence, without reliance on any experimental databeyond the sequence itself. As used herein, “Kabat numbering” refers tothe numbering system set forth by Kabat et al., U.S. Dept. of Health andHuman Services, “Sequence of Proteins of Immunological Interest” (1983).Unless otherwise specified, references to the numbering of specificamino acid residue positions in an antibody variable region areaccording to the Kabat numbering system.

The polypeptide sequences of the sequence listing (i.e., SEQ ID NOs 3,4, 5, 6, 7, 8, 9, etc.) are not numbered according to the Kabatnumbering system. However, it is well within the ordinary skill of onein the art to convert the numbering of the sequences of the SequenceListing to Kabat numbering.

“Framework” or “FR” refers to variable domain residues other thanhypervariable region (HVR) residues. The FR of a variable domaingenerally consists of four FR domains: FR1, FR2, FR3, and FR4.Accordingly, the HVR and FR sequences generally appear in the followingsequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3 (L3)-FR4.

The “class” of an antibody refers to the type of constant domain orconstant region possessed by its heavy chain. There are five majorclasses of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of thesemay be further divided into subclasses (isotypes), e.g., IgG₁, IgG₂,IgG₃, IgG₄, IgA₁, and IgA₂. The heavy chain constant domains thatcorrespond to the different classes of immunoglobulins are called α, δ,ε, γ, and μ, respectively.

The term “Fc region” or “Fc domain” herein is used to define aC-terminal region of an immunoglobulin heavy chain that contains atleast a portion of the constant region. The term includes nativesequence Fc regions and variant Fc regions. Although the boundaries ofthe Fc region of an IgG heavy chain might vary slightly, the human IgGheavy chain Fc region is usually defined to extend from Cys226, or fromPro230, to the carboxyl-terminus of the heavy chain. However, theC-terminal lysine (Lys447) of the Fc region may or may not be present.Unless otherwise specified herein, numbering of amino acid residues inthe Fc region or constant region is according to the EU numberingsystem, also called the EU index, as described in Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md., 1991.

A “region equivalent to the Fc region of an immunoglobulin” is intendedto include naturally occurring allelic variants of the Fc region of animmunoglobulin as well as variants having alterations which producesubstitutions, additions, or deletions but which do not decreasesubstantially the ability of the immunoglobulin to mediate effectorfunctions (such as antibody-dependent cell-mediated cytotoxicity). Forexample, one or more amino acids can be deleted from the N-terminus orC-terminus of the Fc region of an immunoglobulin without substantialloss of biological function. Such variants can be selected according togeneral rules known in the art so as to have minimal effect on activity(see, e.g., Bowie et al., Science 247, 1306-10 (1990)).

A “modification promoting heterodimerization” is a manipulation of thepeptide backbone or the post-translational modifications of apolypeptide, e.g. an immunoglobulin heavy chain, that reduces orprevents the association of the polypeptide with an identicalpolypeptide to form a homodimer. A modification promotingheterodimerization as used herein particularly includes separatemodifications made to each of two polypeptides desired to form a dimer,wherein the modifications are complementary to each other so as topromote association of the two polypeptides. For example, a modificationpromoting heterodimerization may alter the structure or charge of one orboth of the polypeptides desired to form a dimer so as to make theirassociation sterically or electrostatically favorable, respectively.Heterodimerization occurs between two non-identical polypeptides, suchas two immunoglobulin heavy chains wherein further immunoconjugatecomponents fused to each of the heavy chains (e.g. effector moiety) arenot the same. In the immunoconjugates of the present invention, themodification promoting heterodimerization is in the heavy chain(s),specifically in the Fc region, of an immunoglobulin molecule. In someembodiments the modification promoting heterodimerziation comprises anamino acid mutation, specifically an amino acid substitution. In aparticular embodiment, the modification promoting heterodimerizationcomprises a separate amino acid mutation, specifically an amino acidsubstitution, in each of the two immunoglobulin heavy chains.

The term “effector functions” when used in reference to antibodies referto those biological activities attributable to the Fc region of anantibody, which vary with the antibody isotype. Examples of antibodyeffector functions include: C1q binding and complement dependentcytotoxicity (CDC), Fc receptor binding, antibody-dependentcell-mediated cytotoxicity (ADCC), antibody-dependent cellularphagocytosis (ADCP), cytokine secretion, immune complex-mediated antigenuptake by antigen presenting cells, down regulation of cell surfacereceptors (e.g. B cell receptor), and B cell activation.

As used herein, the term “effector cells” refers to a population oflymphocytes that display effector moiety receptors, e.g. cytokinereceptors, and/or Fc receptors on their surface through which they bindan effector moiety, e.g. a cytokine, and/or an Fc region of an antibodyand contribute to the destruction of target cells, e.g. tumor cells.Effector cells may for example mediate cytotoxic or phagocytic effects.Effector cells include, but are not limited to, effector T cells such asCD8⁺ cytotoxic T cells, CD4⁺ helper T cells, γδ T cells, NK cells,lymphokine-activated killer (LAK) cells and macrophages/monocytes.Depending on their receptor expression pattern there may be differentsubsets of effector cells, i.e. (a) cells that express receptors for aparticular effector moiety but no Fc receptors and are stimulated by theimmunoconjugates but not the antibodies of the invention (e.g. T cells,expressing IL-2 receptors); (b) cells that express Fc receptors but noreceptors for a particular effector moiety and are stimulated by theantibodies but not the immunoconjugates of the invention; and (c) cellsthat express both Fc receptors and receptors for a particular effectormoiety and are simultaneously stimulated by the antibodies and theimmunoconjugates of the invention (e.g. NK cells, expressing FcγIIIreceptors and IL-2 receptors).

As used herein, the terms “engineer, engineered, engineering,” areconsidered to include any manipulation of the peptide backbone or thepost-translational modifications of a naturally occurring or recombinantpolypeptide or fragment thereof. Engineering includes modifications ofthe amino acid sequence, of the glycosylation pattern, or of the sidechain group of individual amino acids, as well as combinations of theseapproaches. “Engineering”, particularly with the prefix “glyco-”, aswell as the term “glycosylation engineering” includes metabolicengineering of the glycosylation machinery of a cell, including geneticmanipulations of the oligosaccharide synthesis pathways to achievealtered glycosylation of glycoproteins expressed in cells. Furthermore,glycosylation engineering includes the effects of mutations and cellenvironment on glycosylation. In one embodiment, the glycosylationengineering is an alteration in glycosyltransferase activity. In aparticular embodiment, the engineering results in alteredglucosaminyltransferase activity and/or fucosyltransferase activity.Glycosylation engineering can be used to obtain a “host cell havingincreased GnTIII activity” (e.g. a host cell that has been manipulatedto express increased levels of one or more polypeptides havingβ(1,4)-N-acetylglucosaminyltransferase III (GnTIII) activity), a “hostcell having increased ManII activity” (e.g. a host cell that has beenmanipulated to express increased levels of one or more polypeptideshaving α-mannosidase II (ManII) activity), or a “host cell havingdecreased α(1,6) fucosyltransferase activity” (e.g. a host cell that hasbeen manipulated to express decreased levels of α(1,6)fucosyltransferase).

The term “amino acid mutation” as used herein is meant to encompassamino acid substitutions, deletions, insertions, and modifications. Anycombination of substitution, deletion, insertion, and modification canbe made to arrive at the final construct, provided that the finalconstruct possesses the desired characteristics, e.g., reduced bindingto an Fc receptor. Amino acid sequence deletions and insertions includeamino- and/or carboxy-terminal deletions and insertions of amino acids.Particular amino acid mutations are amino acid substitutions. For thepurpose of altering e.g. the binding characteristics of an Fc region,non-conservative amino acid substitutions, i.e. replacing one amino acidwith another amino acid having different structural and/or chemicalproperties, are particularly preferred. Amino acid substitutions includereplacement by non-naturally occurring amino acids or by naturallyoccurring amino acid derivatives of the twenty standard amino acids(e.g. 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine,5-hydroxylysine). Amino acid mutations can be generated using genetic orchemical methods well known in the art. Genetic methods may includesite-directed mutagenesis, PCR, gene synthesis and the like. It iscontemplated that methods of altering the side chain group of an aminoacid by methods other than genetic engineering, such as chemicalmodification, may also be useful. Various designations may be usedherein to indicate the same amino acid mutation. For example, asubstitution from proline at position 329 of the Fc region to glycinecan be indicated as 329G, G329, G₃₂₉, P329G, or Pro329Gly.

“Percent (%) amino acid sequence identity” with respect to a referencepolypeptide sequence is defined as the percentage of amino acid residuesin a candidate sequence that are identical with the amino acid residuesin the reference polypeptide sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor aligning sequences, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.For purposes herein, however, % amino acid sequence identity values aregenerated using the sequence comparison computer program ALIGN-2. TheALIGN-2 sequence comparison computer program was authored by Genentech,Inc., and the source code has been filed with user documentation in theU.S. Copyright Office, Washington D.C., 20559, where it is registeredunder U.S. Copyright Registration No. TXU510087. The ALIGN-2 program ispublicly available from Genentech, Inc., South San Francisco, Calif., ormay be compiled from the source code. The ALIGN-2 program should becompiled for use on a UNIX operating system, including digital UNIXV4.0D. All sequence comparison parameters are set by the ALIGN-2 programand do not vary. In situations where ALIGN-2 is employed for amino acidsequence comparisons, the % amino acid sequence identity of a givenamino acid sequence A to, with, or against a given amino acid sequence B(which can alternatively be phrased as a given amino acid sequence Athat has or comprises a certain % amino acid sequence identity to, with,or against a given amino acid sequence B) is calculated as follows:100 times the fraction X/Ywhere X is the number of amino acid residues scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofA and B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A. Unless specifically stated otherwise, all % aminoacid sequence identity values used herein are obtained as described inthe immediately preceding paragraph using the ALIGN-2 computer program.

The terms “host cell,” “host cell line,” and “host cell culture” areused interchangeably and refer to cells into which exogenous nucleicacid has been introduced, including the progeny of such cells. Hostcells include “transformants” and “transformed cells,” which include theprimary transformed cell and progeny derived therefrom without regard tothe number of passages. Progeny may not be completely identical innucleic acid content to a parent cell, but may contain mutations. Mutantprogeny that have the same function or biological activity as screenedor selected for in the originally transformed cell are included herein.A host cell is any type of cellular system that can be used to generatethe antibodies and immunoconjugates used for the present invention. Inone embodiment, the host cell is engineered to allow the production ofan antibody with modified oligosaccharides. In certain embodiments, thehost cells have been manipulated to express increased levels of one ormore polypeptides having β(1,4)-N-acetylglucosaminyltransferase III(GnTIII) activity. In certain embodiments the host cells have beenfurther manipulated to express increased levels of one or morepolypeptides having α-mannosidase II (ManII) activity. Host cellsinclude cultured cells, e.g. mammalian cultured cells, such as CHOcells, BHK cells, NS0 cells, SP2/0 cells, YO myeloma cells, P3X63 mousemyeloma cells, PER cells, PER.C6 cells or hybridoma cells, yeast cells,insect cells, and plant cells, to name only a few, but also cellscomprised within a transgenic animal, transgenic plant or cultured plantor animal tissue.

As used herein, the term “polypeptide having GnTIII activity” refers topolypeptides that are able to catalyze the addition of aN-acetylglucosamine (GlcNAc) residue in β-1,4 linkage to the β-linkedmannoside of the trimannosyl core of N-linked oligosaccharides. Thisincludes fusion polypeptides exhibiting enzymatic activity similar to,but not necessarily identical to, an activity ofβ(1,4)-N-acetylglucosaminyltransferase III, also known asβ-1,4-mannosyl-glycoprotein 4-beta-N-acetylglucosaminyl-transferase (EC2.4.1.144), according to the Nomenclature Committee of the InternationalUnion of Biochemistry and Molecular Biology (NC-IUBMB), as measured in aparticular biological assay, with or without dose dependency. In thecase where dose dependency does exist, it need not be identical to thatof GnTIII, but rather substantially similar to the dose-dependency in agiven activity as compared to the GnTIII (i.e. the candidate polypeptidewill exhibit greater activity or not more than about 25-fold less and,preferably, not more than about ten-fold less activity, and mostpreferably, not more than about three-fold less activity relative to theGnTIII). In certain embodiments the polypeptide having GnTIII activityis a fusion polypeptide comprising the catalytic domain of GnTIII andthe Golgi localization domain of a heterologous Golgi residentpolypeptide. Particularly, the Golgi localization domain is thelocalization domain of mannosidase II or GnTI, most particularly thelocalization domain of mannosidase II. Alternatively, the Golgilocalization domain is selected from the group consisting of: thelocalization domain of mannosidase I, the localization domain of GnTII,and the localization domain of α1,6 core fucosyltransferase. Methods forgenerating such fusion polypeptides and using them to produce antibodieswith increased effector functions are disclosed in WO2004/065540, U.S.Provisional Pat. Appl. No. 60/495,142 and U.S. Pat. Appl. Publ. No.2004/0241817, the entire contents of which are expressly incorporatedherein by reference.

As used herein, the term “Golgi localization domain” refers to the aminoacid sequence of a Golgi resident polypeptide which is responsible foranchoring the polypeptide to a location within the Golgi complex.Generally, localization domains comprise amino terminal “tails” of anenzyme.

As used herein, the term “polypeptide having ManII activity” refers topolypeptides that are able to catalyze the hydrolysis of the terminal1,3- and 1,6-linked α-D-mannose residues in the branchedGlcNAcMan₅GlcNAc₂ mannose intermediate of N-linked oligosaccharides.This includes polypeptides exhibiting enzymatic activity similar to, butnot necessarily identical to, an activity of Golgi α-mannosidase II,also known as mannosyl oligosaccharide 1,3-1,6-α-mannosidase II (EC3.2.1.114), according to the Nomenclature Committee of the InternationalUnion of Biochemistry and Molecular Biology (NC-IUBMB).

An “activating Fc receptor” is an Fc receptor that following engagementby an Fc region of an antibody elicits signaling events that stimulatethe receptor-bearing cell to perform effector functions. Activating Fcreceptors include FcγRIIIa (CD16a), FcγRI (CD64), FcγRIIa (CD32), andFcαRI (CD89).

Antibody-dependent cell-mediated cytotoxicity (ADCC) is an immunemechanism leading to the lysis of antibody-coated target cells by immuneeffector cells. The target cells are cells to which antibodies orfragments thereof comprising an Fc region specifically bind, generallyvia the protein part that is N-terminal to the Fc region. As usedherein, the term “increased/reduced ADCC” is defined as either anincrease/reduction in the number of target cells that are lysed in agiven time, at a given concentration of antibody in the mediumsurrounding the target cells, by the mechanism of ADCC defined above,and/or a reduction/increase in the concentration of antibody, in themedium surrounding the target cells, required to achieve the lysis of agiven number of target cells in a given time, by the mechanism of ADCC.The increase/reduction in ADCC is relative to the ADCC mediated by thesame antibody produced by the same type of host cells, using the samestandard production, purification, formulation and storage methods(which are known to those skilled in the art), but that has not beenengineered. For example the increase in ADCC mediated by an antibodyproduced by host cells engineered to have an altered pattern ofglycosylation (e.g. to express the glycosyltransferase, GnTIII, or otherglycosyltransferases) by the methods described herein, is relative tothe ADCC mediated by the same antibody produced by the same type ofnon-engineered host cells.

By “antibody having increased/reduced antibody dependent cell-mediatedcytotoxicity (ADCC)” is meant an antibody having increased/reduced ADCCas determined by any suitable method known to those of ordinary skill inthe art. One accepted in vitro ADCC assay is as follows:

-   -   1) the assay uses target cells that are known to express the        target antigen recognized by the antigen-binding region of the        antibody;    -   2) the assay uses human peripheral blood mononuclear cells        (PBMCs), isolated from blood of a randomly chosen healthy donor,        as effector cells;    -   3) the assay is carried out according to following protocol:    -   i) the PBMCs are isolated using standard density centrifugation        procedures and are suspended at 5×10⁶ cells/ml in RPMI cell        culture medium;    -   ii) the target cells are grown by standard tissue culture        methods, harvested from the exponential growth phase with a        viability higher than 90%, washed in RPMI cell culture medium,        labeled with 100 micro-Curies of ⁵¹Cr, washed twice with cell        culture medium, and resuspended in cell culture medium at a        density of 10⁵ cells/ml;    -   iii) 100 microliters of the final target cell suspension above        are transferred to each well of a 96-well microtiter plate;    -   iv) the antibody is serially-diluted from 4000 ng/ml to 0.04        ng/ml in cell culture medium and 50 microliters of the resulting        antibody solutions are added to the target cells in the 96-well        microtiter plate, testing in triplicate various antibody        concentrations covering the whole concentration range above;    -   v) for the maximum release (MR) controls, 3 additional wells in        the plate containing the labeled target cells, receive 50        microliters of a 2% (V/V) aqueous solution of non-ionic        detergent (Nonidet, Sigma, St. Louis), instead of the antibody        solution (point iv above);    -   vi) for the spontaneous release (SR) controls, 3 additional        wells in the plate containing the labeled target cells, receive        50 microliters of RPMI cell culture medium instead of the        antibody solution (point iv above);    -   vii) the 96-well microtiter plate is then centrifuged at 50×g        for 1 minute and incubated for 1 hour at 4° C.;    -   viii) 50 microliters of the PBMC suspension (point i above) are        added to each well to yield an effector:target cell ratio of        25:1 and the plates are placed in an incubator under 5% CO₂        atmosphere at 37° C. for 4 hours;    -   ix) the cell-free supernatant from each well is harvested and        the experimentally released radioactivity (ER) is quantified        using a gamma counter;    -   x) the percentage of specific lysis is calculated for each        antibody concentration according to the formula        (ER-MR)/(MR-SR)×100, where ER is the average radioactivity        quantified (see point ix above) for that antibody concentration,        MR is the average radioactivity quantified (see point ix above)        for the MR controls (see point v above), and SR is the average        radioactivity quantified (see point ix above) for the SR        controls (see point vi above);    -   4) “increased/reduced ADCC” is defined as either an        increase/reduction in the maximum percentage of specific lysis        observed within the antibody concentration range tested above,        and/or a reduction/increase in the concentration of antibody        required to achieve one half of the maximum percentage of        specific lysis observed within the antibody concentration range        tested above. The increase/reduction in ADCC is relative to the        ADCC, measured with the above assay, mediated by the same        antibody, produced by the same type of host cells, using the        same standard production, purification, formulation and storage        methods, which are known to those skilled in the art, but that        has not been engineered.

As used herein, “combination” (and grammatical variations thereof suchas “combine” or “combining”) encompasses combinations of animmunoconjugate and an antibody according to the invention wherein theimmunoconjugate and the antibody are in the same or in differentcontainers, in the same or in different pharmaceutical formulations,administered together or separately, administered simultaneously orsequentially, in any order, and administered by the same or by differentroutes, provided that the immunoconjugate and the antibody cansimultaneously exert their biological effects in the body, i.e.simultaneously stimulate effector cells. For example “combining” animmunoconjugate and an antibody according to the invention may meanfirst administering the immunoconjugate in a particular pharmaceuticalformulation, followed by administration of the antibody in anotherpharmaceutical formulation, or vice versa.

An “effective amount” of an agent refers to the amount that is necessaryto result in a physiological change in the cell or tissue to which it isadministered.

A “therapeutically effective amount” of an agent, e.g. a pharmaceuticalcomposition, refers to an amount effective, at dosages and for periodsof time necessary, to achieve the desired therapeutic or prophylacticresult. A therapeutically effective amount of an agent for exampleeliminates, decreases, delays, minimizes or prevents adverse effects ofa disease. A therapeutically effective amount of a combination ofseveral active ingredients may be a therapeutically effective amount ofeach of the active ingredients. Alternatively, to reduce the sideeffects caused by the treatment, a therapeutically effective amount of acombination of several active ingredients may be amounts of theindividual active ingredients that are effective to produce an additive,or a superadditive or synergistic effect, and that in combination aretherapeutically effective, but which may be sub-therapeutic amounts ofone or several of the active ingredients if they were used alone.

An “individual” or “subject” is a mammal. Mammals include, but are notlimited to, domesticated animals (e.g. cows, sheep, cats, dogs, andhorses), primates (e.g. humans and non-human primates such as monkeys),rabbits, and rodents (e.g. mice and rats). Particularly, the individualor subject is a human.

The term “pharmaceutical composition” refers to a preparation which isin such form as to permit the biological activity of an activeingredient contained therein to be effective, and which contains noadditional components which are unacceptably toxic to a subject to whichthe formulation would be administered.

A “pharmaceutically acceptable carrier” refers to an ingredient in apharmaceutical formulation, other than an active ingredient, which isnontoxic to a subject. A pharmaceutically acceptable carrier includes,but is not limited to, a buffer, excipient, stabilizer, or preservative.

As used herein, “treatment” (and grammatical variations thereof such as“treat” or “treating”) refers to clinical intervention in an attempt toalter the natural course of a disease in the individual being treated,and can be performed either for prophylaxis or during the course ofclinical pathology. Desirable effects of treatment include, but are notlimited to, preventing occurrence or recurrence of disease, alleviationof symptoms, diminishment of any direct or indirect pathologicalconsequences of the disease, preventing metastasis, decreasing the rateof disease progression, amelioration or palliation of the disease state,and remission or improved prognosis. In some embodiments, combinationsof the invention are used to delay development of a disease or to slowthe progression of a disease.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,combination therapy, contraindications and/or warnings concerning theuse of such therapeutic products.

Immunoconjugates

Immunoconjugates useful in the present invention are polypeptidemolecules that comprise an effector moiety and an antibody engineered tohave reduced effector function, as compared to a correspondingnon-engineered antibody.

Immunoconjugates can be prepared either by chemically conjugating theeffector moiety to the antibody, or by expressing the effector moietyand the antibody as a fusion protein (see, e.g. Nakamura and Kubo,Cancer 80, 2650-2655 (1997); and Becker et al., Proc Natl Acad Sci USA93, 7826-7831 (1996)). For use in the present invention,immunoconjugates expressed as fusion proteins are generally preferred.Accordingly, in certain embodiments the effector moiety shares an amino-or carboxy-terminal peptide bond with the antibody (i.e. theimmunoconjugate is a fusion protein). In such immunoconjugates, aneffector moiety may for example be fused to an immunoglobulin heavy orlight chain. Particularly useful in the present invention areimmunoconjugates comprising a full-length IgG class antibody,particularly a full-length IgG₁ sub-class antibody.

In one embodiment, the effector moiety is a single-chain effectormoiety. In one embodiment the effector moiety is a cytokine. Theantibodies and effector moieties of the immunoconjugate include thosethat are described in detail herein above and below. The antibody of theimmunoconjugate can be directed against a variety of target molecules(e.g. an antigenic determinant on a protein molecule expressed on atumor cell or tumor stroma). Non-limiting examples of antibodies aredescribed herein. Particularly useful immunoconjugates as describedherein typically exhibit one or more of the following properties: highspecificity of action, reduced toxicity, good produceability and/orimproved stability, particularly as compared to immunoconjugates ofdifferent configurations targeting the same antigenic determinants andcarrying the same effector moieties. Particular immunoconjugates for usein the present invention are further described in PCT publication numberWO 2012/146628, the entire contents of which are incorporated herein byreference.

Immunoconjugate Formats

The immunoconjugates described in PCT publication number WO 2012/146628comprise not more than one effector moiety. Accordingly, in a particularembodiment, the immunoconjugate for use in the present inventioncomprises not more than one effector moiety. In a particular embodiment,the effector moiety is a single chain effector moiety. The antibodycomprised in the immunoconjugates according to the invention isparticularly a full-length IgG class antibody, more particularly afull-length IgG₁ sub-class antibody. In one embodiment the antibody ishuman. In other embodiments, the antibody is humanized or chimeric. Inone embodiment, the antibody comprises a human Fc region, moreparticularly a human IgG Fc region, most particularly a human IgG₁ Fcregion. The antibodies useful in the invention may comprise a human Iggamma-1 heavy chain constant region, as set forth in SEQ ID NO: 124(i.e. the antibodies are of human IgG₁ subclass).

In one embodiment the effector moiety shares an amino- orcarboxy-terminal peptide bond with the antibody. In one embodiment, theimmunoconjugate essentially consists of an effector moiety and anantibody, particularly an IgG class antibody, more particularly an IgG₁sub-class antibody, joined by one or more peptide linkers. In a specificembodiment the effector moiety is fused at its amino-terminal amino acidto the carboxy-terminal amino acid of one of the immunoglobulin heavychains, optionally through a peptide linker.

In certain embodiments, particularly where the immunoconjugate comprisesonly a single effector moiety, the antibody comprises in the Fc region amodification promoting heterodimerization of two non-identicalimmunoglobulin heavy chains. The site of most extensive protein-proteininteraction between the two polypeptide chains of a human IgG Fc regionis in the CH3 domain of the Fc region. Thus, in one embodiment saidmodification is in the CH3 domain of the Fc region. In a specificembodiment said modification is a knob-into-hole modification,comprising a knob modification in one of the immunoglobulin heavy chainsand a hole modification in the other one of the immunoglobulin heavychains. The knob-into-hole technology is described e.g. in U.S. Pat.Nos. 5,731,168; 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996)and Carter, J Immunol Meth 248, 7-15 (2001). Generally, the methodinvolves introducing a protuberance (“knob”) at the interface of a firstpolypeptide and a corresponding cavity (“hole”) in the interface of asecond polypeptide, such that the protuberance can be positioned in thecavity so as to promote heterodimer formation and hinder homodimerformation. Protuberances are constructed by replacing small amino acidside chains from the interface of the first polypeptide with larger sidechains (e.g. tyrosine or tryptophan). Compensatory cavities of identicalor similar size to the protuberances are created in the interface of thesecond polypeptide by replacing large amino acid side chains withsmaller ones (e.g. alanine or threonine). The protuberance and cavitycan be made by altering the nucleic acid encoding the polypeptides, e.g.by site-specific mutagenesis, or by peptide synthesis. In a specificembodiment a knob modification comprises the amino acid substitutionT366W in one of the two immunoglobulin heavy chains, and the holemodification comprises the amino acid substitutions T366S, L368A andY407V in the other one of the two immunoglobulin heavy chains (Kabatnumbering). In a further specific embodiment, immunoglobulin heavy chaincomprising the knob modification additionally comprises the amino acidsubstitution S354C, and the immunoglobulin heavy chain comprising thehole modification additionally comprises the amino acid substitutionY349C. Introduction of these two cysteine residues results in formationof a disulfide bridge between the two heavy chains, further stabilizingthe dimer (Carter, J Immunol Methods 248, 7-15 (2001)).

In a particular embodiment the effector moiety is joined to thecarboxy-terminal amino acid of the immunoglobulin heavy chain comprisingthe knob modification.

In an alternative embodiment a modification promoting heterodimerizationof two non-identical polypeptide chains comprises a modificationmediating electrostatic steering effects, e.g. as described in PCTpublication WO 2009/089004. Generally, this method involves replacementof one or more amino acid residues at the interface of the twopolypeptide chains by charged amino acid residues so that homodimerformation becomes electrostatically unfavorable but heterodimerizationelectrostatically favorable.

An Fc region confers to the immunoconjugate favorable pharmacokineticproperties, including a long serum half-life which contributes to goodaccumulation in the target tissue and a favorable tissue-blooddistribution ratio. At the same time it may, however, lead toundesirable targeting of the immunoconjugate to cells expressing Fcreceptors rather than to the preferred antigen-bearing cells. Moreover,the co-activation of Fc receptor signaling pathways may lead to cytokinerelease which, in combination with the effector moiety and the longhalf-life of the immunoconjugate, results in excessive activation ofcytokine receptors and severe side effects upon systemic administration.In line with this, conventional IgG-IL-2 immunoconjugates have beendescribed to be associated with infusion reactions (see e.g. King etal., J Clin Oncol 22, 4463-4473 (2004)).

Accordingly, the antibody comprised in the immunoconjugate is engineeredto have reduced effector function, as compared to a correspondingnon-engineered antibody. In particular embodiments, the reduced effectorfunction is reduced binding to an activating Fc receptor. In one suchembodiment the antibody comprises in its Fc region one or more aminoacid mutation that reduces the binding affinity of the immunoconjugateto an activating Fc receptor. Typically, the same one or more amino acidmutation is present in each of the two immunoglobulin heavy chains. Inone embodiment said amino acid mutation reduces the binding affinity ofthe immunoconjugate to the activating Fc receptor by at least 2-fold, atleast 5-fold, or at least 10-fold. In embodiments where there is morethan one amino acid mutation that reduces the binding affinity of theimmunoconjugate to the activating Fc receptor, the combination of theseamino acid mutations may reduce the binding affinity of theimmunoconjugate to the activating Fc receptor by at least 10-fold, atleast 20-fold, or even at least 50-fold. In one embodiment theimmunoconjugate comprising an engineered antibody exhibits less than20%, particularly less than 10%, more particularly less than 5% of thebinding affinity to an activating Fc receptor as compared to animmunoconjugate comprising a non-engineered antibody. In a specificembodiment the activating Fc receptor is an Fcγ receptor, morespecifically an FcγRIIIa, FcγRI or FcγRIIa receptor. Preferably, bindingto each of these receptors is reduced. In some embodiments bindingaffinity to a complement component, specifically binding affinity toC1q, is also reduced. In one embodiment binding affinity to neonatal Fcreceptor (FcRn) is not reduced. Substantially similar binding to FcRn,i.e. preservation of the binding affinity of the antibody to saidreceptor, is achieved when the antibody (or the immunoconjugatecomprising said antibody) exhibits greater than about 70% of the bindingaffinity of a non-engineered form of the antibody (or theimmunoconjugate comprising said non-engineered form of the antibody) toFcRn. Antibodies, or immunoconjugates comprising said antibodies, mayexhibit greater than about 80% and even greater than about 90% of suchaffinity. In one embodiment the amino acid mutation is an amino acidsubstitution. In one embodiment the antibody, particularly a human IgG₁sub-class antibody, comprises an amino acid substitution at positionP329 of the immunoglobulin heavy chain (Kabat numbering). In a morespecific embodiment the amino acid substitution is P329A or P329G,particularly P329G. In one embodiment the antibody comprises a furtheramino acid substitution at a position selected from 5228, E233, L234,L235, N297 and P331 of the immunoglobulin heavy chain. In a morespecific embodiment the further amino acid substitution is S228P, E233P,L234A, L235A, L235E, N297A, N297D or P331S. In a particular embodimentthe antibody comprises amino acid substitutions at positions P329, L234and L235 of the immunoglobulin heavy chain (Kabat numbering). In a moreparticular embodiment the antibody comprises the amino acidsubstitutions L234A, L235A and P329G (LALA P329G) in the immunoglobulinheavy chain. This combination of amino acid substitutions almostparticularly efficiently abolishes Fcγ receptor binding of a human IgGmolecule, and hence reduces effector function includingantibody-dependent cell-mediated cytotoxicity (ADCC), as described inPCT publication no. WO 2012/130831, incorporated herein by reference inits entirety. WO 2012/130831 also describes methods of preparing suchmutant antibodies and methods for determining its properties such as Fcreceptor binding or effector functions.

Mutant antibodies can be prepared by amino acid deletion, substitution,insertion or modification using genetic or chemical methods well knownin the art. Genetic methods may include site-specific mutagenesis of theencoding DNA sequence, PCR, gene synthesis, and the like. The correctnucleotide changes can be verified for example by sequencing.

Binding to Fc receptors can be easily determined e.g. by ELISA, or bySurface Plasmon Resonance (SPR) using standard instrumentation such as aBIAcore instrument (GE Healthcare), and Fc receptors such as may beobtained by recombinant expression. A suitable such binding assay isdescribed herein. Alternatively, binding affinity of antibodies orimmunoconjugates comprising an antibody for Fc receptors may beevaluated using cell lines known to express particular Fc receptors,such as NK cells expressing FcγIIIa receptor.

In some embodiments the antibody of the immunoconjugate is engineered tohave reduced effector function, particularly reduced ADCC, as comparedto a non-engineered antibody. Effector function of an antibody, or animmunoconjugate comprising an antibody, can be measured by methods knownin the art. A suitable assay for measuring ADCC is described herein.Other examples of in vitro assays to assess ADCC activity of a moleculeof interest are described in U.S. Pat. No. 5,500,362; Hellstrom et al.Proc Natl Acad Sci USA 83, 7059-7063 (1986) and Hellstrom et al., ProcNatl Acad Sci USA 82, 1499-1502 (1985); U.S. Pat. No. 5,821,337;Bruggemann et al., J Exp Med 166, 1351-1361 (1987). Alternatively,non-radioactive assays methods may be employed (see, for example, ACTI™non-radioactive cytotoxicity assay for flow cytometry (CellTechnology,Inc. Mountain View, Calif.); and CytoTox 96® non-radioactivecytotoxicity assay (Promega, Madison, Wis.)). Useful effector cells forsuch assays include peripheral blood mononuclear cells (PBMC) andNatural Killer (NK) cells. Alternatively, or additionally, ADCC activityof the molecule of interest may be assessed in vivo, e.g. in a animalmodel such as that disclosed in Clynes et al., Proc Natl Acad Sci USA95, 652-656 (1998).

In some embodiments binding of the antibody to a complement component,specifically to C1q, is altered. Accordingly, in some embodimentswherein the antibody is engineered to have reduced effector function,said reduced effector function includes reduced CDC. C1q binding assaysmay be carried out to determine whether the immunoconjugate is able tobind C1q and hence has CDC activity. See e.g., C1q and C3c binding ELISAin WO 2006/029879 and WO 2005/100402. To assess complement activation, aCDC assay may be performed (see, for example, Gazzano-Santoro et al., JImmunol Methods 202, 163 (1996); Cragg et al., Blood 101, 1045-1052(2003); and Cragg and Glennie, Blood 103, 2738-2743 (2004)).

In some embodiments, the immunoconjugate comprises one or moreproteolytic cleavage sites located between effector moiety and antibody.Components of the immunoconjugate may be linked directly or throughvarious linkers, particularly peptide linkers comprising one or moreamino acids, typically about 2-20 amino acids, that are described hereinor are known in the art. Suitable, non-immunogenic peptide linkersinclude, for example, (G4S)_(n), (SG₄)_(n) or G₄(SG₄)_(n) peptidelinkers, wherein n is generally a number between 1 and 10, typicallybetween 2 and 4.

Antibodies of Immunoconjugates

The antibody of the immunoconjugate of the invention is generally animmunoglobulin molecule that binds to a specific antigenic determinantand is able to direct the entity to which it is attached (e.g. aneffector moiety) to a target site, for example to a specific type oftumor cell or tumor stroma that bears the antigenic determinant. Theimmunoconjugate can bind to antigenic determinants found, for example,on the surfaces of tumor cells, on the surfaces of virus-infected cells,on the surfaces of other diseased cells, free in blood serum, and/or inthe extracellular matrix (ECM). Non-limiting examples of tumor antigensinclude MAGE, MART-1/Melan-A, gp100, Dipeptidyl peptidase IV (DPPIV),adenosine deaminase-binding protein (ADAbp), cyclophilin b, Colorectalassociated antigen (CRC)-0017-1A/GA733, Carcinoembryonic Antigen (CEA)and its immunogenic epitopes CAP-1 and CAP-2, etv6, aml1, ProstateSpecific Antigen (PSA) and its immunogenic epitopes PSA-1, PSA-2, andPSA-3, prostate-specific membrane antigen (PSMA), T-cellreceptor/CD3-zeta chain, MAGE-family of tumor antigens (e.g., MAGE-A1MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9,MAGE-A10, MAGE-A11, MAGE-A12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3),MAGE-Xp4 (MAGE-B4), MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-05),GAGE-family of tumor antigens (e.g., GAGE-1, GAGE-2, GAGE-3, GAGE-4,GAGE-5, GAGE-6, GAGE-7, GAGE-8, GAGE-9), BAGE, RAGE, LAGE-1, NAG, GnT-V,MUM-1, CDK4, tyrosinase, p53, MUC family, HER2/neu, p21ras, RCAS1,α-fetoprotein, E-cadherin, α-catenin, β-catenin and γ-catenin, p120ctn,gp100 Pmel117, PRAME, NY-ESO-1, cdc27, adenomatous polyposis coliprotein (APC), fodrin, Connexin 37, Ig-idiotype, p15, gp75, GM2 and GD2gangliosides, viral products such as human papilloma virus proteins,Smad family of tumor antigens, lmp-1, P1A, EBV-encoded nuclear antigen(EBNA)-1, brain glycogen phosphorylase, SSX-1, SSX-2 (HOM-MEL-40),SSX-1, SSX-4, SSX-5, SCP-1 and CT-7, and c-erbB-2. Non-limiting examplesof viral antigens include influenza virus hemagglutinin, Epstein-Barrvirus LMP-1, hepatitis C virus E2 glycoprotein, HIV gp160, and HIVgp120. Non-limiting examples of ECM antigens include syndecan,heparanase, integrins, osteopontin, link, cadherins, laminin, laminintype EGF, lectin, fibronectin, notch, tenascin, and matrixin. Theimmunoconjugates of the invention can bind to the following specificnon-limiting examples of cell surface antigens: FAP, Her2, EGFR, IGF-1R,CD2 (T-cell surface antigen), CD3 (heteromultimer associated with theTCR), CD22 (B-cell receptor), CD23 (low affinity IgE receptor), CD25(IL-2 receptor a chain), CD30 (cytokine receptor), CD33 (myeloid cellsurface antigen), CD40 (tumor necrosis factor receptor), IL-6R (IL6receptor), CD20, MCSP, c-Met, CUB domain-containing protein-1 (CDCP1),and PDGFβR (β platelet-derived growth factor receptor).

In certain embodiments the antibody is directed to an antigen presentedon a tumor cell or in a tumor cell environment. In a specific embodimentthe antibody is directed to an antigen selected from the group ofFibroblast Activation Protein (FAP), the A1 domain of Tenascin-C (TNCA1), the A2 domain of Tenascin-C (TNC A2), the Extra Domain B ofFibronectin (EDB), Carcinoembryonic Antigen (CEA) andMelanoma-associated Chondroitin Sulfate Proteoglycan (MCSP).

The antibody can be any type of antibody or fragment thereof thatretains specific binding to an antigenic determinant and comprises an Fcregion. In one embodiment the antibody is a full-length antibody.Particularly preferred antibodies are immunoglobulins of the IgG class,specifically of the IgG₁ subclass.

In one embodiment, the immunoconjugate comprises an antibody that isspecific for the A1 and/or the A4 domain of Tenascin (TNC-A1 or TNC-A4or TNC-A1/A4). In a specific embodiment, the antibody of theimmunoconjugate comprises a heavy chain variable region sequence that isat least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identicalto either SEQ ID NO: 8 or SEQ ID NO: 9, or variants thereof that retainfunctionality. In another specific embodiment, the antibody of theimmunoconjugate comprises a light chain variable region sequence that isat least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identicalto either SEQ ID NO: 6 or SEQ ID NO: 7, or variants thereof that retainfunctionality. In a more specific embodiment, the antibody of theimmunoconjugate comprises a heavy chain variable region sequence that isat least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identicalto either SEQ ID NO: 8 or SEQ ID NO: 9 or variants thereof that retainfunctionality, and a light chain variable region sequence that is atleast about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical toeither SEQ ID NO: 6 or SEQ ID NO: 7 or variants thereof that retainfunctionality.

In one embodiment, the immunoconjugate comprises an antibody that isspecific for the A2 domain of Tenascin (TNC-A2). In a specificembodiment, the antibody of the immunoconjugate comprises a heavy chainvariable region sequence that is at least about 80%, 85%, 90%, 95%, 96%,97%, 98%, 99% or 100% identical to a sequence selected from the group ofSEQ ID NO: 5, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO:77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83 and SEQ ID NO: 85, orvariants thereof that retain functionality. In another specificembodiment, the antibody of the immunoconjugate comprises a light chainvariable region sequence that is at least about 80%, 85%, 90%, 95%, 96%,97%, 98%, 99% or 100% identical to a sequence selected from the group ofSEQ ID NO: 3, SEQ ID NO: 4; SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74,SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82 and SEQ IDNO: 84, or variants thereof that retain functionality. In a morespecific embodiment, the antibody of the immunoconjugate comprises aheavy chain variable region sequence that is at least about 80%, 85%,90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selectedfrom the group of SEQ ID NO: 5, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO:75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83 and SEQID NO: 85, or variants thereof that retain functionality, and a lightchain variable region sequence that is at least about 80%, 85%, 90%,95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selected fromthe group of SEQ ID NO: 3, SEQ ID NO: 4; SEQ ID NO: 70, SEQ ID NO: 72,SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO:82 and SEQ ID NO: 84, or variants thereof that retain functionality.

In one embodiment, the immunoconjugate comprises an antibody that isspecific for the Fibroblast Activated Protein (FAP). In a specificembodiment, the antibody of the immunoconjugate comprises a heavy chainvariable region sequence that is at least about 80%, 85%, 90%, 95%, 96%,97%, 98%, 99% or 100% identical to a sequence selected from the groupconsisting of SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO:17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ IDNO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45,SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO:55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ IDNO: 65, SEQ ID NO: 67 and SEQ ID NO: 69, or variants thereof that retainfunctionality. In another specific embodiment, the antibody of theimmunoconjugate comprises a light chain variable region sequence that isat least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identicalto a sequence selected from the group consisting of: SEQ ID NO: 10, SEQID NO: 11, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20,SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO:30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ IDNO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58,SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66 and SEQ IDNO: 68, or variants thereof that retain functionality. In a morespecific embodiment, the antibody of the immunoconjugate comprises aheavy chain variable region sequence that is at least about 80%, 85%,90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selectedfrom the group consisting of SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO:15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ IDNO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43,SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO:53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ IDNO: 63, SEQ ID NO: 65, SEQ ID NO: 67 and SEQ ID NO: 69, or variantsthereof that retain functionality, and a light chain variable regionsequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or 100% identical to a sequence selected from the group consisting of:SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO:18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ IDNO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46,SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO:56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ IDNO: 66 and SEQ ID NO: 68, or variants thereof that retain functionality.In another specific embodiment, the antibody of the immunoconjugatecomprises the heavy chain variable region sequence of SEQ ID NO: 12 andthe light chain variable region sequence of SEQ ID NO: 11. In anotherspecific embodiment, the antibody of the immunoconjugate comprises theheavy chain variable region sequence of SEQ ID NO: 17 and the lightchain variable region sequence of SEQ ID NO: 16. In another specificembodiment, the antibody of the immunoconjugate comprises the heavychain variable region sequence of SEQ ID NO: 47 and the light chainvariable region sequence of SEQ ID NO: 46. In another specificembodiment, the antibody of the immunoconjugate comprises the heavychain variable region sequence of SEQ ID NO: 63 and the light chainvariable region sequence of SEQ ID NO: 62. In another specificembodiment, the antibody of the immunoconjugate comprises the heavychain variable region sequence of SEQ ID NO: 67 and the light chainvariable region sequence of SEQ ID NO: 66. In another specificembodiment, the immunoconjugate of the present invention comprises apolypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%,97%, 98%, 99% or 100% identical to SEQ ID NO: 125 or variants thereofthat retain functionality, a polypeptide sequence that is at least about80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:126 or variants thereof that retain functionality, and a polypeptidesequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or 100% identical to SEQ ID NO: 129 or variants thereof that retainfunctionality. In another specific embodiment, the immunoconjugate ofthe present invention comprises a polypeptide sequence that is at leastabout 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ IDNO: 127 or variants thereof that retain functionality, a polypeptidesequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or 100% identical to SEQ ID NO: 128 or variants thereof that retainfunctionality, and a polypeptide sequence that is at least about 80%,85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 129 orvariants thereof that retain functionality. In another specificembodiment, the immunoconjugate of the present invention comprises apolypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%,97%, 98%, 99% or 100% identical to SEQ ID NO: 130 or variants thereofthat retain functionality, a polypeptide sequence that is at least about80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:131 or variants thereof that retain functionality, and a polypeptidesequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or 100% identical to SEQ ID NO: 132 or variants thereof that retainfunctionality.

In one embodiment, the immunoconjugate comprises an antibody that isspecific for the Melanoma Chondroitin Sulfate Proteoglycan (MCSP). In aspecific embodiment, the antibody of the immunoconjugate comprises aheavy chain variable region sequence that is at least about 80%, 85%,90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of eitherSEQ ID NO: 86 or SEQ ID NO: 122 or variants thereof that retainfunctionality. In another specific embodiment, the antibody of theimmunoconjugate comprises a light chain variable region sequence that isat least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identicalto the sequence of either SEQ ID NO: 87 or SEQ ID NO: 123 or variantsthereof that retain functionality. In a more specific embodiment, theantibody of the immunoconjugate comprises a heavy chain variable regionsequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or 100% identical to the sequence of either SEQ ID NO: 86 or SEQ ID NO:122, or variants thereof that retain functionality, and a light chainvariable region sequence that is at least about 80%, 85%, 90%, 95%, 96%,97%, 98%, 99% or 100% identical to the sequence of either SEQ ID NO: 87or SEQ ID NO: 123, or variants thereof that retain functionality. In amore specific embodiment, the antibody of the immunoconjugate comprisesa heavy chain variable region sequence that is at least about 80%, 85%,90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ IDNO: 86, and a light chain variable region sequence that is at leastabout 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to thesequence of SEQ ID NO: 87. In another specific embodiment, the antibodyof the immunoconjugate comprises a heavy chain variable region sequencethat is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%identical to the sequence of SEQ ID NO: 122, and a light chain variableregion sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%,98%, 99% or 100% identical to the sequence of SEQ ID NO: 123.

In one embodiment, the immunoconjugate comprises an antibody that isspecific for the Carcinoembryonic Antigen (CEA). In a specificembodiment, the antibody of the immunoconjugate comprises a heavy chainvariable region sequence that is at least about 80%, 85%, 90%, 95%, 96%,97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 114 or avariant thereof that retains functionality. In another specificembodiment, the antibody of the immunoconjugate comprises a light chainvariable region sequence that is at least about 80%, 85%, 90%, 95%, 96%,97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 115 or avariant thereof that retains functionality. In a more specificembodiment, the antibody of the immunoconjugate comprises a heavy chainvariable region sequence that is at least about 80%, 85%, 90%, 95%, 96%,97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 114, or avariant thereof that retains functionality, and a light chain variableregion sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%,98%, 99% or 100% identical to the sequence of SEQ ID NO: 115, or avariant thereof that retains functionality. In another specificembodiment, the immunoconjugate of the present invention comprises apolypeptide sequence that is at least about 80%, 85%, 90%, 95%, 96%,97%, 98%, 99% or 100% identical to SEQ ID NO: 136 or variants thereofthat retain functionality, a polypeptide sequence that is at least about80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:137 or variants thereof that retain functionality, and a polypeptidesequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or 100% identical to SEQ ID NO: 138 or variants thereof that retainfunctionality.

Immunoconjugates according to the invention include those that comprisesequences that are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%,99%, or 100% identical to the sequences set forth in SEQ ID NOs 3-87,108-132 and 136-138, including functional fragments or variants thereof.The immunoconjugates according to the invention also encompassesantibodies comprising sequences of SEQ ID NOs 3-127 with conservativeamino acid substitutions. It is understood that in the sequences of SEQID NOs 126, 128, 131, 134 and 137, the sequence of the sequence of themutant IL-2 described herein (see SEQ ID NO: 2) may be replaced by thesequence of human IL-2 (see SEQ ID NO: 1).

Effector Moieties of Immunoconjugates

The effector moieties for use in the invention are generallypolypeptides that influence cellular activity, for example, throughsignal transduction pathways. Accordingly, the effector moiety of theimmunoconjugate useful in the invention can be associated withreceptor-mediated signaling that transmits a signal from outside thecell membrane to modulate a response within the cell. For example, aneffector moiety of the immunoconjugate can be a cytokine. In aparticular embodiment, the effector moiety is a single-chain effectormoiety as defined herein. In one embodiment, the effector moiety,typically a single-chain effector moiety, of the immunoconjugateaccording to the invention is a cytokine selected from the groupconsisting of: IL-2, GM-CSF, IFN-α, and IL-12. In one embodiment theeffector moiety is IL-2. In another embodiment, the single-chaineffector moiety of the immunoconjugate is a cytokine selected from thegroup consisting of: IL-8, MIP-la, and TGF-β.

In one embodiment, the effector moiety, particularly a single-chaineffector moiety, of the immunoconjugate is IL-2. In a specificembodiment, the IL-2 effector moiety can elicit one or more of thecellular responses selected from the group consisting of: proliferationin an activated T lymphocyte cell, differentiation in an activated Tlymphocyte cell, cytotoxic T cell (CTL) activity, proliferation in anactivated B cell, differentiation in an activated B cell, proliferationin a natural killer (NK) cell, differentiation in a NK cell, cytokinesecretion by an activated T cell or an NK cell, and NK/lymphocyteactivated killer (LAK) antitumor cytotoxicity. In certain embodiments,the IL-2 effector moiety is a mutant IL-2 effector moiety comprising atleast one amino acid mutation that reduces or abolishes the affinity ofthe mutant IL-2 effector moiety to the α-subunit of the IL-2 receptor(also known as CD25) but preserves the affinity of the mutant IL-2effector moiety to the intermediate-affinity IL-2 receptor (consistingof the β- and γ-subunits of the IL-2 receptor), compared to thenon-mutated IL-2 effector moiety. In one embodiment the amino acidmutations are amino acid substitutions. In a specific embodiment, themutant IL-2 effector moiety comprises one, two or three amino acidsubstitutions at one, two or three position(s) selected from thepositions corresponding to residue 42, 45, and 72 of human IL-2 (SEQ IDNO: 1). In a more specific embodiment, the mutant IL-2 effector moietycomprises three amino acid substitutions at the positions correspondingto residue 42, 45 and 72 of human IL-2. In an even more specificembodiment, the mutant IL-2 effector moiety is human IL-2 comprising theamino acid substitutions F42A, Y45A and L72G. In one embodiment themutant IL-2 effector moiety additionally comprises an amino acidmutation at a position corresponding to position 3 of human IL-2, whicheliminates the O-glycosylation site of IL-2. Particularly saidadditional amino acid mutation is an amino acid substitution replacing athreonine residue by an alanine residue. The sequence of a quadruplemutant (QM) IL-2 comprising the amino acid substitutions T3A, F42A, Y45Aand L72G is shown in SEQ ID NO: 2. Suitable mutant IL-2 molecules aredescribed in more detail in PCT publication number WO 2012/107417.

Mutant IL-2 molecules useful as effector moieties in theimmunoconjugates can be prepared by deletion, substitution, insertion ormodification using genetic or chemical methods well known in the art.Genetic methods may include site-specific mutagenesis of the encodingDNA sequence, PCR, gene synthesis, and the like. The correct nucleotidechanges can be verified for example by sequencing. In this regard, thenucleotide sequence of native IL-2 has been described by Taniguchi etal. (Nature 302, 305-10 (1983)) and nucleic acid encoding human IL-2 isavailable from public depositories such as the American Type CultureCollection (Rockville Md.). An exemplary sequence of human IL-2 is shownin SEQ ID NO: 1. Substitution or insertion may involve natural as wellas non-natural amino acid residues. Amino acid modification includeswell known methods of chemical modification such as the addition orremoval of glycosylation sites or carbohydrate attachments, and thelike.

In one embodiment, the effector moiety, particularly a single-chaineffector moiety, of the immunoconjugate is GM-CSF. In a specificembodiment, the GM-CSF effector moiety can elicit proliferation and/ordifferentiation in a granulocyte, a monocyte or a dendritic cell. In oneembodiment, the effector moiety, particularly a single-chain effectormoiety, of the immunoconjugate is IFN-α. In a specific embodiment, theIFN-α effector moiety can elicit one or more of the cellular responsesselected from the group consisting of: inhibiting viral replication in avirus-infected cell, and upregulating the expression of majorhistocompatibility complex I (MHC I). In another specific embodiment,the IFN-α effector moiety can inhibit proliferation in a tumor cell. Inone embodiment, the effector moiety, particularly a single-chaineffector moiety, of the immunoconjugate is IL-12. In a specificembodiment, the IL-12 effector moiety can elicit one or more of thecellular responses selected from the group consisting of: proliferationin a NK cell, differentiation in a NK cell, proliferation in a T cell,and differentiation in a T cell. In one embodiment, the effector moiety,particularly a single-chain effector moiety, of the immunoconjugate isIL-8. In a specific embodiment, the IL-8 effector moiety can elicitchemotaxis in neutrophils. In one embodiment, the effector moiety,particularly a single-chain effector moiety, of the immunoconjugate, isMIP-1α. In a specific embodiment, the MIP-1α effector moiety can elicitchemotaxis in monocytes and T lymphocyte cells. In one embodiment, theeffector moiety, particularly a single-chain effector moiety, of theimmunoconjugate is MIP-1β. In a specific embodiment, the MIP-1β effectormoiety can elicit chemotaxis in monocytes and T lymphocyte cells. In oneembodiment, the effector moiety, particularly a single-chain effectormoiety, of the immunoconjugate is TGF-β. In a specific embodiment, theTGF-β effector moiety can elicit one or more of the cellular responsesselected from the group consisting of: chemotaxis in monocytes,chemotaxis in macrophages, upregulation of IL-1 expression in activatedmacrophages, and upregulation of IgA expression in activated B cells.

Antibodies for Combination with the Immunconjugates

According to the invention, antibodies for combination with theimmunoconjugates are engineered to have increased effector function.Antibodies useful in the present invention for combination with theimmunoconjugates include antibodies or antibody fragments that bind to aspecific antigenic determinant, for example a specific tumor cellantigen, and comprise an Fc region. In certain embodiments the antibodyis directed to an antigen presented on a tumor cell. Particular targetantigens of the antibodies useful in the present invention includeantigens expressed on the surface of tumor cells, including, but notlimited to, cell surface receptors such as epidermal growth factorreceptor (EGFR), insulin-like growth factor receptors (IGFR) andplatelet-derived growth factor receptors (PDGFR), prostate specificmembrane antigen (PSMA), carcinoembryonic antigen (CEA), dipeptidylpeptidase IV (CD26, DPPIV), FAP, HER2/neu, HER-3, E-cadherin, CD20,melanoma-associated chondroitin sulfate proteoglycan (MCSP), c-Met, CUBdomain-containing protein-1 (CDCP1), and squamous cell carcinoma antigen(SCCA).

In a specific embodiment the antibody is directed to an antigen selectedfrom the group of CD20, Epidermal Growth Factor Receptor (EGFR), HER2,HER3, Insulin-like Growth Factor 1 Receptor (IGF-1R), CarcinoembryonicAntigen (CEA), c-Met, CUB domain-containing protein-1 (CDCP1), andMelanoma-associated Chondroitin Sulfate Proteoglycan (MCSP). In oneembodiment, the antibody a multispecific antibody directed to two ormore antigens selected from the group of CD20, Epidermal Growth FactorReceptor (EGFR), HER2, HER3, Insulin-like Growth Factor 1 Receptor(IGF-1R), Carcinoembryonic Antigen (CEA), c-Met, CUB domain-containingprotein-1 (CDCP1), and Melanoma-associated Chondroitin SulfateProteoglycan (MCSP).

Specific anti-CD20 antibodies useful in the present invention arehumanized, IgG-class Type II anti-CD20 antibodies, having the bindingspecificity of the murine B-Ly1 antibody (Poppema and Visser, BiotestBulletin 3, 131-139 (1987)). Particularly useful is a humanized,IgG-class Type II anti-CD20 antibody, comprising

-   -   a) in the heavy chain variable domain a CDR1 of SEQ ID NO: 88, a        CDR2 of SEQ ID NO: 89, and a CDR3 of SEQ ID NO: 90, and    -   b) in the light chain variable domain a CDR1 of SEQ ID NO: 91, a        CDR2 of SEQ ID NO: 92, and a CDR3 of SEQ ID NO: 93.

Particularly, the heavy chain variable region framework regions (FRs)FR1, FR2, and FR3 of said antibody are human FR sequences encoded by theVH1_10 human germ-line sequence, the heavy chain variable region FR4 ofsaid antibody is a human FR sequence encoded by the JH4 human germ-linesequence, the light chain variable region FRs FR1, FR2, and FR3 of saidantibody are human FR sequences encoded by the VK_2_40 human germ-linesequence, and the light chain variable region FR4 of said antibody is ahuman FR sequence encoded by the JK4 human germ-line sequence.

A more particular anti-CD20 antibody which is useful in the presentinvention comprises the heavy chain variable domain of SEQ ID NO: 94 andthe light chain variable domain of SEQ ID NO: 95.

Such anti-CD20 antibodies are described in WO 2005/044859, which isincorporated herein by reference in its entirety.

Specific anti-EGFR antibodies useful in the present invention arehumanized, IgG-class antibodies, having the binding specificity of therat ICR62 antibody (Modjtahedi et al., Br J Cancer 67, 247-253 (1993)).Particularly useful is a humanized, IgG-class anti-EGFR antibody,comprising

-   -   a) in the heavy chain variable domain a CDR1 of SEQ ID NO: 96, a        CDR2 of SEQ ID NO: 97, and a CDR3 of SEQ ID NO: 98, and    -   b) in the light chain variable domain a CDR1 of SEQ ID NO: 99, a        CDR2 of SEQ ID NO: 100, and a CDR3 of SEQ ID NO: 101.

A more particular anti-EGFR antibody which is useful in the inventioncomprises the heavy chain variable domain of SEQ ID NO: 102 and thelight chain variable domain of SEQ ID NO: 103.

Such anti-EGFR antibodies are described in WO 2006/082515 and WO2008/017963, each of which is incorporated herein by reference in itsentirety.

Other suitable humanized IgG-class anti-EGFR antibodies useful for theinvention include cetuximab/IMC-C225 (Erbitux®, described in Goldsteinet al., Clin Cancer Res 1, 1311-1318 (1995)), panitumumab/ABX-EGF(Vectibix®, described in Yang et al., Cancer Res 59, 1236-1243 (1999),Yang et al., Critical Reviews in Oncology/Hematology 38, 17-23 (2001)),nimotuzumab/h-R3 (TheraCim®, described in Mateo et al., Immunotechnology3, 71-81 (1997); Crombet-Ramos et al., Int J Cancer 101, 567-575 (2002),Boland & Bebb, Expert Opin Biol Ther 9, 1199-1206 (2009)), matuzumab/EMD72000 (described in Bier et al., Cancer Immunol Immunother 46, 167-173(1998), Kim, Curr Opin Mol Ther 6, 96-103 (2004)), and zalutumumab/2F8(described in Bleeker et al., J Immunol 173, 4699-4707 (2004), Lammertsvan Bueren, PNAS 105, 6109-6114 (2008)).

Specific anti-IGF-1R antibodies useful in the present invention aredescribed in WO 2005/005635 and WO 2008/077546, the entire content ofeach of which is incorporated herein by reference, and inhibit thebinding of insulin-like growth factor-1 (IGF-1) and insulin-like growthfactor-2 (IGF-2) to insulin-like growth factor-1 receptor (IGF-1R).

The anti-IGF-1R antibodies useful for the invention are preferablymonoclonal antibodies and, in addition, chimeric antibodies (humanconstant domain), humanized antibodies and especially preferably fullyhuman antibodies. Particular anti-IGF-1R antibodies useful for theinvention bind to human IGF-1R in competition to antibody 18, i.e. theybind to the same epitope of IGF-1R as antibody 18, which is described inWO 2005/005635. Particular anti-IGF-1R antibodies are furthercharacterized by an affinity to IGF-1R of 10⁻⁸ M (K_(D)) or less,particularly of about 10⁻⁹ to 10⁻¹³ M, and preferably show no detectableconcentration-dependent inhibition of insulin binding to the insulinreceptor.

Particular anti-IGF-1R antibodies useful for the invention comprisecomplementarity determining regions (CDRs) having the followingsequences:

-   -   a) an antibody heavy chain comprising as CDRs CDR1, CDR2 and        CDR3 of SEQ ID NO: 104 or 106;    -   b) an antibody light chain comprising as CDRs CDR1, CDR2 and        CDR3 of SEQ ID NO: 105 or 107.

Particularly, the anti-IGF-1R antibodies useful for the inventioncomprise an antibody heavy chain variable domain amino acid sequence ofSEQ ID NO: 104 and an antibody light chain variable domain amino acidsequence of SEQ ID NO: 105, or an antibody heavy chain variable domainamino acid sequence of SEQ ID NO: 106 and an antibody light chainvariable domain amino acid sequence of SEQ ID NO: 107.

Particular anti-IGF-1R antibodies useful for the invention areobtainable from the hybridoma cell lines <IGF-1R> HUMAB-Clone 18 and<IGF-1R> HUMAB-Clone 22, which are deposited with Deutsche Sammlung vonMikroorganismen and Zellkulturen GmbH (DSMZ), Germany, under depositionnumbers DSM ACC 2587 and DSM ACC 2594, respectively.

Other suitable anti-IGF-1R antibodies useful for the invention are e.g.the fully human IgG₁ mAb cixutumumab/IMC-A12 (described in Burtrum etal., Cancer Res 63, 8912-21 (2003); Rowinsky et al., Clin Cancer Res 13,5549s-5555s (2007), the fully human IgGl mAb AMG-479 (described inBeltran et al., Mol Cancer Ther 8, 1095-1105 (2009); Tolcher et al., JClin Oncol 27, 5800-7 (2009)), the humanized IgG₁ mAb MK-0646/h7C10(described in Goetsch et al., Int J Cancer 113, 316-28 (2005); Broussaset al., Int J Cancer 124, 2281-93 (2009); Hidalgo et al., J Clin Oncol26, abstract 3520 (2008); Atzori et al., J Clin Oncol 26, abstract 3519(2008)), the humanized IgG₁ mAb AVE1642 (described in Descamps et al.,Br J Cancer 100, 366-9 (2009); Tolcher et al., J Clin Oncol 26, abstract3582 (2008); Moreau et al., Blood 110, abstract 1166 (2007); Maloney etal., Cancer Res 63, 5073-83 (2003)), the fully human IgG₂ mAbfigitumumab/CP-751,871 (Cohen et al., Clin Cancer Res 11, 2063-73(2005); Haluska et al., Clin Cancer Res 13, 5834-40 (2007); Lacy et al.,J Clin Oncol 26, 3196-203 (2008); Gualberto & Karp, Clin Lung Cancer 10,273-80 (2009), the fully human IgG₁ mAb SCH-717454 (described in WO2008/076257 or Kolb et al., Pediatr Blood Cancer 50, 1190-7 (2008)), the2.13.2. mAb (described in U.S. Pat. No. 7,037,498 (WO 2002/053596)) orthe fully human IgG₄ mAb BIIB022.

Specific anti-CEA antibodies useful in the present invention arehumanized, IgG-class antibodies, having the binding specificity of themurine PR1A3 antibody (Richman and Bodmer, Int J Cancer 39, 317-328(1987)). Particularly useful is a humanized, IgG-class anti-CEAantibody, comprising

-   -   a) in the heavy chain variable domain a CDR1 of SEQ ID NO: 108,        a CDR2 of SEQ ID NO: 109, and a CDR3 of SEQ ID NO: 110, and    -   b) in the light chain variable domain a CDR1 of SEQ ID NO: 111,        a CDR2 of SEQ ID NO: 112, and a CDR3 of SEQ ID NO: 113.

A more particular anti-CEA antibody which is useful in the inventioncomprises the heavy chain variable domain of SEQ ID NO: 114 and thelight chain variable domain of SEQ ID NO: 115.

Such anti-CEA antibodies are described in PCT publication number WO2011/023787, which is incorporated herein by reference in its entirety.

Specific anti-HER3 antibodies that are useful in the present inventionare humanized, IgG-class antibodies, such as the Mab 205.10.1, Mab205.10.2 and Mab 205.10.3, particularly Mab 205.10.2, described in PCTpublication number WO 2011/076683. Particularly useful is a humanized,IgG-class anti-HER3 antibody, comprising

-   -   a) in the heavy chain variable domain a CDR1 of SEQ ID NO: 139,        a CDR2 of SEQ ID NO: 140, and a CDR3 of SEQ ID NO: 141, and    -   b) in the light chain variable domain a CDR1 of SEQ ID NO: 143,        a CDR2 of SEQ ID NO: 144, and a CDR3 of SEQ ID NO: 145.

A more particular anti-HER3 antibody which is useful in the inventioncomprises the heavy chain variable domain of SEQ ID NO: 142 and thelight chain variable domain of SEQ ID NO: 146.

Specific anti-CDCP1-antibodies that are useful in the present inventionare humanized, IgG-class antibodies derived from the CUB4 antibody(deposition number DSM ACC 2551 (DSMZ), as described in PCT publicationnumber WO 2011/023389.

Exemplary anti-MCSP antibodies that can be used in the present inventionare described e.g. in European patent application number EP 11178393.2.Particularly useful is a humanized, IgG-class anti-MCSP antibody,comprising

-   -   a) in the heavy chain variable domain a CDR1 of SEQ ID NO: 116,        a CDR2 of SEQ ID NO: 117, and a CDR3 of SEQ ID NO: 118, and    -   b) in the light chain variable domain a CDR1 of SEQ ID NO: 119,        a CDR2 of SEQ ID NO: 120, and a CDR3 of SEQ ID NO: 121.

A more particular anti-MCSP antibody which is useful in the inventioncomprises the heavy chain variable domain of SEQ ID NO: 122 and thelight chain variable domain of SEQ ID NO: 123.

In one embodiment the antibody is a full-length antibody of theIgG-class. In a particular embodiment, the antibody is an IgG₁ antibody.In one embodiment, the antibody comprises a human Fc region, moreparticularly a human IgG Fc region, most particularly a human IgG₁ Fcregion. The antibodies useful in the invention, such as the anti-IGF-1R,anti-EGFR and anti-CD20 antibodies described above, may comprise a humanIg gamma-1 heavy chain constant region, as set forth in SEQ ID NO: 124(i.e. the antibodies are of human IgG₁ subclass).

The antibodies useful in the present invention are engineered to haveincreased effector function, as compared to a correspondingnon-engineered antibody. In one embodiment the antibody engineered tohave increased effector function has at least 2-fold, at least 10-foldor even at least 100-fold increased effector function, compared to acorresponding non-engineered antibody. The increased effector functioncan include, but is not limited to, one or more of the following:increased Fc receptor binding, increased C1q binding and complementdependent cytotoxicity (CDC), increased antibody-dependent cell-mediatedcytotoxicity (ADCC), increased antibody-dependent cellular phagocytosis(ADCP), increased cytokine secretion, increased immune complex-mediatedantigen uptake by antigen-presenting cells, increased binding to NKcells, increased binding to macrophages, increased binding to monocytes,increased binding to polymorphonuclear cells, increased direct signalinginducing apoptosis, increased crosslinking of target-bound antibodies,increased dendritic cell maturation, or increased T cell priming.

In one embodiment the increased effector function one or more selectedfrom the group of increased Fc receptor binding, increased CDC,increased ADCC, increased ADCP, and increased cytokine secretion. In oneembodiment the increased effector function is increased binding to anactivating Fc receptor. In one such embodiment the binding affinity tothe activating Fc receptor is increased at least 2-fold, particularly atleast 10-fold, compared to the binding affinity of a correspondingnon-engineered antibody. In a specific embodiment the activating Fcreceptor is selected from the group of FcγRIIIa, FcγRI, and FcγRIIa. Inone embodiment the activating Fc receptor is FcγRIIIa, particularlyhuman FcγRIIIa. In another embodiment the increased effector function isincreased ADCC. In one such embodiment the ADCC is increased at least10-fold, particularly at least 100-fold, compared to the ADCC mediatedby a corresponding non-engineered antibody. In yet another embodimentthe increased effector function is increased binding to an activating Fcreceptor and increased ADCC.

Increased effector function can be measured by methods known in the art.A suitable assay for measuring ADCC is described herein. Other examplesof in vitro assays to assess ADCC activity of a molecule of interest aredescribed in U.S. Pat. No. 5,500,362; Hellstrom et al. Proc Natl AcadSci USA 83, 7059-7063 (1986) and Hellstrom et al., Proc Natl Acad SciUSA 82, 1499-1502 (1985); U.S. Pat. No. 5,821,337; Bruggemann et al., JExp Med 166, 1351-1361 (1987). Alternatively, non-radioactive assaysmethods may be employed (see, for example, ACTI™ non-radioactivecytotoxicity assay for flow cytometry (CellTechnology, Inc. MountainView, Calif.); and CytoTox 96® non-radioactive cytotoxicity assay(Promega, Madison, Wis.)). Useful effector cells for such assays includeperipheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells.Alternatively, or additionally, ADCC activity of the molecule ofinterest may be assessed in vivo, e.g. in a animal model such as thatdisclosed in Clynes et al., Proc Natl Acad Sci USA 95, 652-656 (1998).Binding to Fc receptors can be easily determined e.g. by ELISA, or bySurface Plasmon Resonance (SPR) using standard instrumentation such as aBIAcore instrument (GE Healthcare), and Fc receptors such as may beobtained by recombinant expression. According to a particularembodiment, binding affinity to an activating Fc receptor is measured bysurface plasmon resonance using a BIACORE® T100 machine (GE Healthcare)at 25° C. Alternatively, binding affinity of antibodies for Fc receptorsmay be evaluated using cell lines known to express particular Fcreceptors, such as NK cells expressing FcγIIIa receptor. C1q bindingassays may also be carried out to determine whether the antibody is ableto bind C1q and hence has CDC activity. See e.g., C1q and C3c bindingELISA in WO 2006/029879 and WO 2005/100402. To assess complementactivation, a CDC assay may be performed (see, for example,Gazzano-Santoro et al., J Immunol Methods 202, 163 (1996); Cragg et al.,Blood 101, 1045-1052 (2003); and Cragg and Glennie, Blood 103, 2738-2743(2004)).

Increased effector function may result e.g. from glycoengineering of theFc region or the introduction of amino acid mutations in the Fc regionof the antibody. In one embodiment the antibody is engineered byintroduction of one or more amino acid mutations in the Fc region. In aspecific embodiment the amino acid mutations are amino acidsubstitutions. In an even more specific embodiment the amino acidsubstitutions are at positions 298, 333, and/or 334 of the Fc region (EUnumbering of residues). Further suitable amino acid mutations aredescribed e.g. in Shields et al., J Biol Chem 9(2), 6591-6604 (2001);U.S. Pat. No. 6,737,056; WO 2004/063351 and WO 2004/099249. Mutant Fcregions can be prepared by amino acid deletion, substitution, insertionor modification using genetic or chemical methods well known in the art.Genetic methods may include site-specific mutagenesis of the encodingDNA sequence, PCR, gene synthesis, and the like. The correct nucleotidechanges can be verified for example by sequencing.

In another embodiment the antibody is engineered by modification of theglycosylation in the Fc region. In a specific embodiment the antibody isengineered to have an increased proportion of non-fucosylatedoligosaccharides in the Fc region as compared to a non-engineeredantibody. An increased proportion of non-fucosylated oligosaccharides inthe Fc region of an antibody results in the antibody having increasedeffector function, in particular increased ADCC.

In a more specific embodiment, at least about 20%, about 25%, about 30%,about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%,or about 100%, preferably at least about 50%, more preferably at leastabout 70%, of the N-linked oligosaccharides in the Fc region of theantibody are non-fucosylated. The non-fucosylated oligosaccharides maybe of the hybrid or complex type.

In another specific embodiment the antibody is engineered to have anincreased proportion of bisected oligosaccharides in the Fc region ascompared to a non-engineered antibody. In a more specific embodiment, atleast about 10%, about 15%, about 20%, about 25%, about 30%, about 35%,about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about100%, preferably at least about 50%, more preferably at least about 70%,of the N-linked oligosaccharides in the Fc region of the antibody arebisected. The bisected oligosaccharides may be of the hybrid or complextype.

In yet another specific embodiment the antibody is engineered to have anincreased proportion of bisected, non-fucosylated oligosaccharides inthe Fc region, as compared to a non-engineered antibody. In a morespecific embodiment, at least about 10%, about 15%, about 20%, about25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%,about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about90%, about 95%, or about 100%, preferably at least about 15%, morepreferably at least about 25%, at least about 35% or at least about 50%,of the N-linked oligosaccharides in the Fc region of the antibody arebisected, non-fucosylated. The bisected, non-fucosylatedoligosaccharides may be of the hybrid or complex type.

The oligosaccharide structures in the antibody Fc region can be analysedby methods well known in the art, e.g. by MALDI TOF mass spectrometry asdescribed in Umana et al., Nat Biotechnol 17, 176-180 (1999) or Ferraraet al., Biotechn Bioeng 93, 851-861 (2006). The percentage ofnon-fucosylated oligosaccharides is the amount of oligosaccharideslacking fucose residues, relative to all oligosaccharides attached toAsn 297 (e.g. complex, hybrid and high mannose structures) andidentified in an N-glycosidase F treated sample by MALDI TOF MS. Asn 297refers to the asparagine residue located at about position 297 in the Fcregion (EU numbering of Fc region residues); however, Asn297 may also belocated about ±3 amino acids upstream or downstream of position 297,i.e., between positions 294 and 300, due to minor sequence variations inantibodies. The percentage of bisected, or bisected non-fucosylated,oligosaccharides is determined analogously.

In one embodiment the antibody is engineered to have modifiedglycosylation in the Fc region, as compared to a non-engineeredantibody, by producing the antibody in a host cell having alteredactivity of one or more glycosyltransferase. Glycosyltransferasesinclude β(1,4)-N-acetylglucosaminyltransferase III (GnTIII),β(1,4)-galactosyltransferase (GalT),β(1,2)-N-acetylglucosaminyltransferase I (GnTI),β(1,2)-N-acetylglucosaminyltransferase II (GnTII) andα(1,6)-fucosyltransferase. In a specific embodiment the antibody isengineered to have an increased proportion of non-fucosylatedoligosaccharides in the Fc region, as compared to a non-engineeredantibody, by producing the antibody in a host cell having increasedβ(1,4)-N-acetylglucosaminyltransferase III (GnTIII) activity. In an evenmore specific embodiment the host cell additionally has increasedα-mannosidase II (ManII) activity. The glycoengineering methodology thatcan be used for engineering antibodies useful for the present inventionhas been described in greater detail in Umana et al., Nat Biotechnol 17,176-180 (1999); Ferrara et al., Biotechn Bioeng 93, 851-861 (2006); WO99/54342 (U.S. Pat. No. 6,602,684; EP 1071700); WO 2004/065540 (U.S.Pat. Appl. Publ. No. 2004/0241817; EP 1587921), WO 03/011878 (U.S. Pat.Appl. Publ. No. 2003/0175884), the entire content of each of which isincorporated herein by reference in its entirety. Antibodiesglycoengineered using this methodology are referred to as GLYCOMABsherein.

Generally, any type of cultured cell line, including the cell linesdiscussed herein, can be used to generate cell lines for the productionof anti-TNC A2 antibodies with altered glycosylation pattern. Particularcell lines include CHO cells, BHK cells, NS0 cells, SP2/0 cells, YOmyeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells orhybridoma cells, and other mammalian cells. In certain embodiments, thehost cells have been manipulated to express increased levels of one ormore polypeptides having β(1,4)-N-acetylglucosaminyltransferase III(GnTIII) activity. In certain embodiments the host cells have beenfurther manipulated to express increased levels of one or morepolypeptides having α-mannosidase II (ManII) activity. In a specificembodiment, the polypeptide having GnTIII activity is a fusionpolypeptide comprising the catalytic domain of GnTIII and the Golgilocalization domain of a heterologous Golgi resident polypeptide.Particularly, said Golgi localization domain is the Golgi localizationdomain of mannosidase II. Methods for generating such fusionpolypeptides and using them to produce antibodies with increasedeffector functions are disclosed in Ferrara et al., Biotechn Bioeng 93,851-861 (2006) and WO2004/065540, the entire contents of which areexpressly incorporated herein by reference.

The host cells which contain the coding sequence of an antibody usefulfor the invention and/or the coding sequence of polypeptides havingglycosyltransferase activity, and which express the biologically activegene products may be identified e.g. by DNA-DNA or DNA-RNAhybridization; the presence or absence of “marker” gene functions;assessing the level of transcription as measured by the expression ofthe respective mRNA transcripts in the host cell; or detection of thegene product as measured by immunoassay or by its biologicalactivity—methods which are well known in the art. GnTIII or Man IIactivity can be detected e.g. by employing a lectin which binds tobiosynthetis products of GnTIII or ManII, respectively. An example forsuch a lectin is the E₄-PHA lectin which binds preferentially tooligosaccharides containing bisecting GlcNAc. Biosynthesis products(i.e. specific oligosaccharide structures) of polypeptides having GnTIIIor ManII activity can also be detected by mass spectrometric analysis ofoligosaccharides released from glycoproteins produced by cellsexpressing said polypeptides. Alternatively, a functional assay whichmeasures the increased effector function, e.g. increased Fc receptorbinding, mediated by antibodies produced by the cells engineered withthe polypeptide having GnTIII or ManII activity may be used.

In another embodiment the antibody is engineered to have an increasedproportion of non-fucosylated oligosaccharides in the Fc region, ascompared to a non-engineered antibody, by producing the antibody in ahost cell having decreased α(1,6)-fucosyltransferase activity. A hostcell having decreased α(1,6)-fucosyltransferase activity may be a cellin which the α(1,6)-fucosyltransferase gene has been disrupted orotherwise deactivated, e.g. knocked out (see Yamane-Ohnuki et al.,Biotech Bioeng 87, 614 (2004); Kanda et al., Biotechnol Bioeng, 94(4),680-688 (2006); Niwa et al., J Immunol Methods 306, 151-160 (2006)).

Other examples of cell lines capable of producing defucosylatedantibodies include Lec13 CHO cells deficient in protein fucosylation(Ripka et al., Arch Biochem Biophys 249, 533-545 (1986); US Pat. Appl.No. US 2003/0157108; and WO 2004/056312, especially at Example 11). Theantibodies useful in the present invention can alternatively beglycoengineered to have reduced fucose residues in the Fc regionaccording to the techniques disclosed in EP 1 176 195 A1, WO 03/084570,WO 03/085119 and U.S. Pat. Appl. Pub. Nos. 2003/0115614, 2004/093621,2004/110282, 2004/110704, 2004/132140, U.S. Pat. No. 6,946,292 (Kyowa),e.g. by reducing or abolishing the activity of a GDP-fucose transporterprotein in the host cells used for antibody production.

Glycoengineered antibodies useful in the invention may also be producedin expression systems that produce modified glycoproteins, such as thosetaught in WO 03/056914 (GlycoFi, Inc.) or in WO 2004/057002 and WO2004/024927 (Greenovation).

Recombinant Methods

Methods to produce antibodies and immunoconjugates useful in theinvention are well known in the art, and described for example in WO2012/146628, WO 2005/044859, WO 2006/082515, WO 2008/017963, WO2005/005635, WO 2008/077546, WO 2011/023787, WO 2011/076683, WO2011/023389 and WO 2006/100582. Established methods to producepolyclonal antibodies and monoclonal antibodies are also described,e.g., in Harlow and Lane, “Antibodies, a laboratory manual”, Cold SpringHarbor Laboratory, 1988.

Non-naturally occurring antibodies or fragments thereof can beconstructed using solid phase-peptide synthesis, can be producedrecombinantly (e.g. as described in U.S. Pat. No. 4,816,567) or can beobtained, for example, by screening combinatorial libraries comprisingvariable heavy chains and variable light chains (see e.g. U.S. Pat. No.5,969,108 to McCafferty). For recombinant production of immunoconjugatesand antibodies useful in the invention, one or more polynucleotide(s)encoding said immunoconjugate or antibody is isolated and inserted intoone or more vectors for further cloning and/or expression in a hostcell. Such polynucleotides may be readily isolated and sequenced usingconventional procedures. Methods which are well known to those skilledin the art can be used to construct expression vectors containing thecoding sequence of an antibody or immunoconjugate along with appropriatetranscriptional/translational control signals. These methods include invitro recombinant DNA techniques, synthetic techniques and in vivorecombination/genetic recombination. See, for example, the techniquesdescribed in Maniatis et al., MOLECULAR CLONING: A LABORATORY MANUAL,Cold Spring Harbor Laboratory, N.Y. (1989); and Ausubel et al., CURRENTPROTOCOLS IN MOLECULAR BIOLOGY, Greene Publishing Associates and WileyInterscience, N.Y (1989).

Immunoconjugates useful in the invention may be expressed from a singlepolynucleotide that encodes the entire immunoconjugate or from multiple(e.g., two or more) polynucleotides that are co-expressed. Polypeptidesencoded by polynucleotides that are co-expressed may associate through,e.g., disulfide bonds or other means to form a functionalimmunoconjugate. For example, the light chain portion of an antibody maybe encoded by a separate polynucleotide from the portion of theimmunoconjugate comprising the heavy chain portion of the antibody andthe effector moiety. When coexpressed, the heavy chain polypeptides willassociate with the light chain polypeptides to form the antibody.

Host cells suitable for replicating and for supporting expression ofrecombinant proteins are well known in the art. Such cells may betransfected or transduced as appropriate with the particular expressionvector and large quantities of vector containing cells can be grown forseeding large scale fermenters to obtain sufficient quantities of theproteins, e.g. for clinical applications. Suitable host cells includeprokaryotic microorganisms, such as E. coli, or various eukaryoticcells, such as Chinese hamster ovary cells (CHO), insect cells, or thelike. For example, recombinant proteins may be produced in bacteria inparticular when glycosylation is not needed. After expression, theprotein may be isolated from the bacterial cell paste in a solublefraction and can be further purified. In addition to prokaryotes,eukaryotic microbes such as filamentous fungi or yeast are suitablecloning or expression hosts for protein-encoding vectors, includingfungi and yeast strains whose glycosylation pathways have been“humanized,” resulting in the production of a protein with a partiallyor fully human glycosylation pattern. See Gerngross, Nat Biotech 22,1409-1414 (2004), and Li et al., Nat Biotech 24, 210-215 (2006).Suitable host cells for the expression of (glycosylated) proteins arealso derived from multicellular organisms (invertebrates andvertebrates). Examples of invertebrate cells include plant and insectcells. Numerous baculoviral strains have been identified which may beused in conjunction with insect cells, particularly for transfection ofSpodoptera frugiperda cells. Plant cell cultures can also be utilized ashosts. See e.g. U.S. Pat. Nos. 5,959,177; 6,040,498; 6,420,548;7,125,978, and 6,417,429 (describing PLANTIBODIES™ technology forproducing antibodies in transgenic plants). Vertebrate cells may also beused as hosts. For example, mammalian cell lines that are adapted togrow in suspension may be useful. Other examples of useful mammalianhost cell lines are monkey kidney CV1 line transformed by SV40 (COS-7);human embryonic kidney (HEK) line (293 or 293T cells as described, e.g.,in Graham et al., J Gen Virol 36, 59 (1977)), baby hamster kidney cells(BHK), mouse sertoli cells (TM4 cells as described, e.g., in Mather,Biol Reprod 23, 243-251 (1980)), monkey kidney cells (CV1), Africangreen monkey kidney cells (VERO-76), human cervical carcinoma cells(HELA), canine kidney cells (MDCK), buffalo rat liver cells (BRL 3A),human lung cells (W138), human liver cells (Hep G2), mouse mammary tumorcells (MMT 060562), TRI cells (as described, e.g., in Mather et al.,Annals N.Y. Acad Sci 383, 44-68 (1982)), MRC 5 cells, and FS4 cells.Other useful mammalian host cell lines include Chinese hamster ovary(CHO) cells, including dhfr⁻ CHO cells (Urlaub et al., Proc Natl AcadSci USA 77, 4216 (1980)); and myeloma cell lines such as YO, NS0, P3X63and Sp2/0. For a review of certain mammalian host cell lines suitablefor protein production, see, e.g., Yazaki and Wu, Methods in MolecularBiology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, N.J.), pp.255-268 (2003). Host cells include cultured cells, e.g., mammaliancultured cells, yeast cells, insect cells, bacterial cells and plantcells, to name only a few, but also cells comprised within a transgenicanimal, transgenic plant or cultured plant or animal tissue. In oneembodiment, the host cell is a eukaryotic cell, particularly a mammaliancell, e.g. a Chinese Hamster Ovary (CHO) cell, a human embryonic kidney(HEK) 293 cell, or lymphoid cell (e.g., Y0, NS0, Sp20 cell).

If the antibody and immunoconjugate are intended for human use, chimericforms of antibodies may be used wherein the antibody constant regionsare from a human. A humanized or fully human form of the antibody canalso be prepared in accordance with methods well known in the art (seee.g. U.S. Pat. No. 5,565,332 to Winter). Humanization may be achieved byvarious methods including, but not limited to (a) grafting the non-human(e.g., donor antibody) CDRs onto human (e.g. recipient antibody)framework and constant regions with or without retention of criticalframework residues (e.g. those that are important for retaining goodantigen binding affinity or antibody functions), (b) grafting only thenon-human specificity-determining regions (SDRs or a-CDRs; the residuescritical for the antibody-antigen interaction) onto human framework andconstant regions, or (c) transplanting the entire non-human variabledomains, but “cloaking” them with a human-like section by replacement ofsurface residues. Humanized antibodies and methods of making them arereviewed, e.g., in Almagro and Fransson, Front Biosci 13, 1619-1633(2008), and are further described, e.g., in Riechmann et al., Nature332, 323-329 (1988); Queen et al., Proc Natl Acad Sci USA 86,10029-10033 (1989); U.S. Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and7,087,409; Jones et al., Nature 321, 522-525 (1986); Morrison et al.,Proc Natl Acad Sci 81, 6851-6855 (1984); Morrison and Oi, Adv Immunol44, 65-92 (1988); Verhoeyen et al., Science 239, 1534-1536 (1988);Padlan, Molec Immun 31(3), 169-217 (1994); Kashmiri et al., Methods 36,25-34 (2005) (describing SDR (a-CDR) grafting); Padlan, Mol Immunol 28,489-498 (1991) (describing “resurfacing”); Dall'Acqua et al., Methods36, 43-60 (2005) (describing “FR shuffling”); and Osbourn et al.,Methods 36, 61-68 (2005) and Klimka et al., Br J Cancer 83, 252-260(2000) (describing the “guided selection” approach to FR shuffling).Human antibodies and human variable regions can be produced usingvarious techniques known in the art. Human antibodies are describedgenerally in van Dijk and van de Winkel, Curr Opin Pharmacol 5, 368-74(2001) and Lonberg, Curr Opin Immunol 20, 450-459 (2008). Human variableregions can form part of and be derived from human monoclonal antibodiesmade by the hybridoma method (see e.g. Monoclonal Antibody ProductionTechniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York,1987)). Human antibodies and human variable regions may also be preparedby administering an immunogen to a transgenic animal that has beenmodified to produce intact human antibodies or intact antibodies withhuman variable regions in response to antigenic challenge (see e.g.Lonberg, Nat Biotech 23, 1117-1125 (2005). Human antibodies and humanvariable regions may also be generated by isolating Fv clone variableregion sequences selected from human-derived phage display libraries(see e.g., Hoogenboom et al. in Methods in Molecular Biology 178, 1-37(O'Brien et al., ed., Human Press, Totowa, N.J., 2001); and McCaffertyet al., Nature 348, 552-554; Clackson et al., Nature 352, 624-628(1991)). Phage typically display antibody fragments, either assingle-chain Fv (scFv) fragments or as Fab fragments.

In certain embodiments, the antibodies useful in the present inventionare engineered to have enhanced binding affinity according to, forexample, the methods disclosed in U.S. Pat. Appl. Publ. No.2004/0132066, the entire contents of which are hereby incorporated byreference. The ability of the antibodies useful in the invention to bindto a specific antigenic determinant can be measured either through anenzyme-linked immunosorbent assay (ELISA) or other techniques familiarto one of skill in the art, e.g. surface plasmon resonance technique(analyzed on a BIACORE T100 system) (Liljeblad, et al., Glyco J 17,323-329 (2000)), and traditional binding assays (Heeley, Endocr Res 28,217-229 (2002)).

Antibodies and immunoconjugates prepared as described herein may bepurified by art-known techniques such as high performance liquidchromatography, ion exchange chromatography, gel electrophoresis,affinity chromatography, size exclusion chromatography, and the like.The actual conditions used to purify a particular protein will depend,in part, on factors such as net charge, hydrophobicity, hydrophilicityetc., and will be apparent to those having skill in the art.

Pharmaceutical Compositions

In another aspect the invention provides a pharmaceutical compositioncomprising (a) an immunoconjugate comprising a first antibody engineeredto have reduced effector function and an effector moiety, and (b) asecond antibody engineered to have increased effector function, in apharmaceutically acceptable carrier. These pharmaceutical compositionsmay be used, e.g., in any of the therapeutic methods described below.

Pharmaceutical compositions of an immunoconjugate and an antibody havingincreased effector function as described herein are prepared by mixingsuch immunoconjugate and antibody having the desired degree of puritywith one or more optional pharmaceutically acceptable carriers(Remington's Pharmaceutical Sciences 18th edition, Mack Printing Company(1990)), in the form of lyophilized formulations or aqueous solutions.Pharmaceutically acceptable carriers are generally non-toxic torecipients at the dosages and concentrations employed, and include, butare not limited to: buffers such as phosphate, citrate, and otherorganic acids; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride; benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g. Zn-proteincomplexes); and/or non-ionic surfactants such as polyethylene glycol(PEG). Exemplary pharmaceutically acceptable carriers herein furtherinclude insterstitial drug dispersion agents such as solubleneutral-active hyaluronidase glycoproteins (sHASEGP), for example, humansoluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®,Baxter International, Inc.). Certain exemplary sHASEGPs and methods ofuse, including rHuPH20, are described in US Patent Publication Nos.2005/0260186 and 2006/0104968. In one aspect, a sHASEGP is combined withone or more additional glycosaminoglycanases such as chondroitinases.

Exemplary lyophilized formulations are described in U.S. Pat. No.6,267,958. Aqueous formulations include those described in U.S. Pat. No.6,171,586 and WO2006/044908, the latter formulations including ahistidine-acetate buffer.

The pharmaceutical composition herein may also contain additional activeingredients as necessary for the particular indication being treated,particularly those with complementary activities that do not adverselyaffect each other. For example, if the disease to be treated is cancer,it may be desirable to further provide one or more anti-cancer agents,e.g. a chemotherapeutic agent, an inhibitor of tumor cell proliferation,or an activator of tumor cell apoptosis. Such active ingredients aresuitably present in combination in amounts that are effective for thepurpose intended.

Active ingredients may be entrapped in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization,for example, hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacylate) microcapsules, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences18th edition, Mack Printing Company (1990).

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g. films, or microcapsules.

The compositions to be used for in vivo administration are generallysterile. Sterility may be readily accomplished, e.g., by filtrationthrough sterile filtration membranes.

Methods of Treatment

The combination provided herein of (a) an immunoconjugate comprising afirst antibody engineered to have reduced effector function and aneffector moiety, and (b) a second antibody engineered to have increasedeffector function, may be used in therapeutic methods.

In one aspect, a combination of (a) an immunoconjugate comprising afirst antibody engineered to have reduced effector function and aneffector moiety, and (b) a second antibody engineered to have increasedeffector function, for use as a medicament is provided. In furtheraspects, a combination of (a) an immunoconjugate comprising a firstantibody engineered to have reduced effector function and an effectormoiety, and (b) a second antibody engineered to have increased effectorfunction, for use in treating a disease is provided. In certainembodiments, a combination of (a) an immunoconjugate comprising a firstantibody engineered to have reduced effector function and an effectormoiety, and (b) a second antibody engineered to have increased effectorfunction, for use in a method of treatment is provided. In certainembodiments, the invention provides a combination of (a) animmunoconjugate comprising a first antibody engineered to have reducedeffector function and an effector moiety, and (b) a second antibodyengineered to have increased effector function, for use in a method oftreating an individual having a disease comprising administering to theindividual a therapeutically effective amount of the combination. In onesuch embodiment, the method further comprises administering to theindividual a therapeutically effective amount of at least one additionaltherapeutic agent, e.g., as described below. In further embodiments, theinvention provides a combination of (a) an immunoconjugate comprising afirst antibody engineered to have reduced effector function and aneffector moiety, and (b) a second antibody engineered to have increasedeffector function, for use in stimulating effector cell function. Incertain embodiments, the invention provides a combination of (a) animmunoconjugate comprising a first antibody engineered to have reducedeffector function and an effector moiety, and (b) a second antibodyengineered to have increased effector function, for use in a method ofstimulating effector cell function in an individual comprisingadministering to the individual an effective amount of the combinationto stimulate effector cell function. An “individual” according to any ofthe above embodiments is a mammal, particularly a human. A “disease”according to any of the above embodiments is a disease treatable bystimulation of effector cell function. In certain embodiments thedisease is a cell proliferation disorder, particularly cancer.

In a further aspect, the invention provides for the use of a combinationof (a) an immunoconjugate comprising a first antibody engineered to havereduced effector function and an effector moiety, and (b) a secondantibody engineered to have increased effector function, in themanufacture or preparation of a medicament. In one embodiment, themedicament is for treatment of a disease. In a further embodiment, themedicament is for use in a method of treating a disease comprisingadministering to an individual having the disease a therapeuticallyeffective amount of the medicament. In one such embodiment, the methodfurther comprises administering to the individual a therapeuticallyeffective amount of at least one additional therapeutic agent, e.g., asdescribed below. In a further embodiment, the medicament is forstimulating effector cell function. In a further embodiment, themedicament is for use in a method of stimulating effector cell functionin an individual comprising administering to the individual an amount ofthe medicament effective to stimulate effector cell function. An“individual” according to any of the above embodiments is a mammal,particularly a human. A “disease” according to any of the aboveembodiments is a disease treatable by stimulation of effector cellfunction. In certain embodiments the disease is a cell proliferationdisorder, particularly cancer.

In a further aspect, the invention provides a method for treating adisease. In one embodiment, the method comprises administering to anindividual having such disease a therapeutically effective amount of acombination of (a) an immunoconjugate comprising a first antibodyengineered to have reduced effector function and an effector moiety, and(b) a second antibody engineered to have increased effector function. Inone such embodiment, the method further comprises administering to theindividual a therapeutically effective amount of at least one additionaltherapeutic agent, as described below. An “individual” according to anyof the above embodiments is a mammal, particularly a human. A “disease”according to any of the above embodiments is a disease treatable bystimulation of effector cell function. In certain embodiments thedisease is a cell proliferation disorder, particularly cancer.

In a further aspect, the invention provides a method for stimulatingeffector cell function in an individual. In one embodiment, the methodcomprises administering to the individual an effective amount of acombination of (a) an immunoconjugate comprising a first antibodyengineered to have reduced effector function and an effector moiety, and(b) a second antibody engineered to have increased effector function, tostimulate effector cell function. In one embodiment, an “individual” isa mammal, particularly a human.

In a further aspect, the invention provides pharmaceutical compositioncomprising any of the combinations of (a) an immunoconjugate comprisinga first antibody engineered to have reduced effector function and aneffector moiety, and (b) a second antibody engineered to have increasedeffector function provided herein, e.g., for use in any of the abovetherapeutic methods. In one embodiment, a pharmaceutical compositioncomprises a combination provided herein, of (a) an immunoconjugatecomprising a first antibody engineered to have reduced effector functionand an effector moiety and (b) a second antibody engineered to haveincreased effector function, and a pharmaceutically acceptable carrier.In another embodiment, a pharmaceutical composition comprises any of thecombinations provided herein and at least one additional therapeuticagent, e.g., as described below.

According to any of the above embodiments, the disease is a disordertreatable by stimulation of effector cell function. Combinations of theinvention are useful in treating disease states where stimulation of theimmune system of the host is beneficial, in particular conditions wherean enhanced cellular immune response is desirable. These may includedisease states where the host immune response is insufficient ordeficient. Disease states for which the combinations of the inventioncan be administered comprise, for example, a tumor or infection where acellular immune response would be a critical mechanism for specificimmunity. Specific disease states for which the combinations of thepresent invention can be employed include cancer, specifically renalcell carcinoma or melanoma; immune deficiency, specifically inHIV-positive patients, immunosuppressed patients, chronic infection andthe like. In certain embodiments the disease is a cell proliferationdisorder. In a particular embodiment the disease is cancer, specificallya cancer selected from the group of lung cancer, colorectal cancer,renal cancer, prostate cancer, breast cancer, head and neck cancer,ovarian cancer, brain cancer, lymphoma, leukemia, skin cancer.

Combinations of the invention can be used either alone or together withother agents in a therapy. For instance, a combination of the inventionmay be co-administered with at least one additional therapeutic agent.In certain embodiments, an additional therapeutic agent is ananti-cancer agent, e.g. a chemotherapeutic agent, an inhibitor of tumorcell proliferation, or an activator of tumor cell apoptosis.

Combination therapies as provided herein encompass administration of theantibody and the immunoconjugate together (where the two or moretherapeutic agents are included in the same or separate formulations),and separately, in which case, administration of the antibody can occurprior to, simultaneously, and/or following, administration of theimmunoconjugate, additional therapeutic agent and/or adjuvant.Combinations of the invention can also be combined with radiationtherapy.

A combination of the invention (and any additional therapeutic agent)can be administered by any suitable route, including parenteral,intrapulmonary, and intranasal, and, if desired for local treatment,intralesional administration. Parenteral infusions includeintramuscular, intravenous, intraarterial, intraperitoneal, orsubcutaneous administration. The antibody and the immunconjugate may beadministered by the same or by different routes. Dosing can be by anysuitable route, e.g. by injections, such as intravenous or subcutaneousinjections, depending in part on whether the administration is brief orchronic. Various dosing schedules including but not limited to single ormultiple administrations over various time-points, bolus administration,and pulse infusion are contemplated herein.

Combinations of the invention would be formulated, dosed, andadministered in a fashion consistent with good medical practice. Factorsfor consideration in this context include the particular disorder beingtreated, the particular mammal being treated, the clinical condition ofthe individual patient, the cause of the disorder, the site of deliveryof the agents, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners. Thecombination need not be, but is optionally formulated with one or moreagents currently used to prevent or treat the disorder in question. Theeffective amount of such other agents depends on the amount of antibodyand immunoconjugate present in the formulation, the type of disorder ortreatment, and other factors discussed above. These are generally usedin the same dosages and with administration routes as described herein,or about from 1 to 99% of the dosages described herein, or in any dosageand by any route that is empirically/clinically determined to beappropriate.

For the prevention or treatment of disease, the appropriate dosage of anantibody and immunoconjugate (when used in the combinations of theinvention, optionally together with one or more other additionaltherapeutic agents) will depend on the type of disease to be treated,the type of antibody and immunoconjugate, the severity and course of thedisease, whether the combination is administered for preventive ortherapeutic purposes, previous therapy, the patient's clinical historyand response to the antibody and/or immunoconjugate, and the discretionof the attending physician. The antibody and the immunoconjugate aresuitably administered to the patient at one time or over a series oftreatments.

Depending on the type and severity of the disease, about 1 mg/kg to 15mg/kg (e.g. 0.1 mg/kg-10 mg/kg) of antibody can be an initial candidatedosage for administration to the patient, whether, for example, by oneor more separate administrations, or by continuous infusion. One typicaldaily dosage might range from about 1 mg/kg to 100 mg/kg or more,depending on the factors mentioned above. For repeated administrationsover several days or longer, depending on the condition, the treatmentwould generally be sustained until a desired suppression of diseasesymptoms occurs. One exemplary dosage of the antibody would be in therange from about 0.05 mg/kg to about 10 mg/kg. Thus, one or more dosesof about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg or 10 mg/kg (or any combinationthereof) may be administered to the patient. Such doses may beadministered intermittently, e.g. every week or every three weeks (e.g.such that the patient receives from about two to about twenty, or e.g.about six doses of the antibody). An initial higher loading dose,followed by one or more lower doses may be administered. An exemplarydosing regimen comprises administering an initial loading dose of about4 mg/kg, followed by a weekly maintenance dose of about 2 mg/kg of theantibody. The same considerations with respect to dosage apply to theimmunconjugate to be used in the combinations according to theinvention. However, other dosage regimens may be useful. The progress ofthis therapy is easily monitored by conventional techniques and assays.

Articles of Manufacture

In another aspect of the invention, an article of manufacture containingmaterials useful for the treatment, prevention and/or diagnosis of thedisorders described above is provided. The article of manufacturecomprises one or more container and a label or package insert on orassociated with the container. Suitable containers include, for example,bottles, vials, syringes, IV solution bags, etc. The containers may beformed from a variety of materials such as glass or plastic. Thecontainer holds a composition which is by itself or combined withanother composition effective for treating, preventing and/or diagnosingthe condition and may have a sterile access port (for example thecontainer may be an intravenous solution bag or a vial having a stopperpierceable by a hypodermic injection needle). At least one active agentin the composition is an antibody to be used in the combinations of theinvention. Another active agent is the immunoconjugate to be used in thecombinations of the invention, which may be in the same composition andcontainer like the antibody, or may be provided in a differentcomposition and container. The label or package insert indicates thatthe composition is used for treating the condition of choice.

In one aspect the invention provides a kit intended for the treatment ofa disease, comprising in the same or in separate containers (a) animmunoconjugate comprising a first antibody engineered to have reducedeffector function and an effector moiety, and (b) a second antibodyengineered to have increased effector function, and optionally furthercomprising (c) a package insert comprising printed instructionsdirecting the use of the combined treatment as a method for treating thedisease. Moreover, the kit may comprise (a) a first container with acomposition contained therein, wherein the composition comprises anantibody engineered to have increased effector function; (b) a secondcontainer with a composition contained therein, wherein the compositioncomprises an immunoconjugate comprising an antibody engineered to havereduced effector function and an effector moiety; and optionally (c) athird container with a composition contained therein, wherein thecomposition comprises a further cytotoxic or otherwise therapeuticagent. The kit in this embodiment of the invention may further comprisea package insert indicating that the compositions can be used to treat aparticular condition. Alternatively, or additionally, the kit mayfurther comprise a third (or fourth) container comprising apharmaceutically-acceptable buffer, such as bacteriostatic water forinjection (BWFI), phosphate-buffered saline, Ringer's solution anddextrose solution. It may further include other materials desirable froma commercial and user standpoint, including other buffers, diluents,filters, needles, and syringes.

EXAMPLES

The following are examples of methods and compositions of the invention.It is understood that various other embodiments may be practiced, giventhe general description provided above.

General Methods

Glycoengineereing of the Fc region of an antibody leads to increasedbinding affinity to human FcγRIII receptors, which in turn translatesinto enhanced ADCC induction and anti-tumor efficacy. Human FcγRIIIreceptors are expressed on macrophages, neutrophils, and natural killer(NK), dendritic and γδ T cells. In the mouse, the most widely utilizedspecies for preclinical efficacy testing, murine FcγRIV, the murinehomologue of human FcγRIIIa, is present on marcophages and neutrophilsbut not on NK cells. Therefore, not the full extent of any expectedimproved efficacy with glycoengineered antibodies is reflected in thosemodels. We have generated a mouse transgenic for human FcγRIIIa (CD16a),exhibiting stable human CD16a expression on murine NK cells in blood,lymphoid tissues and tumors. Moreover, the expression level of humanCD16a on unstimulated NK cells in the blood of these transgenic micemirrors that found in human. We also showed that a down-regulation ofhuman FcγRIIIa on the tumor-associated NK cells after antibody therapycorrelates with antitumoral activity. Finally, we showed significantlyimproved efficacy of glycoengineered antibody treatment in tumor modelsusing this new mouse strain as compared to their human CD16-negativelittermates.

Example 1

FaDu Head and Neck Carcinoma Xenograft Model

The FAP-targeted 28H1 IgG-IL2 and untargeted DP47GS IgG-IL2immunoconjugates comprising the IL-2 quadruple mutant (qm) (SEQ ID NOs:125, 126, 129, and SEQ ID NOs: 133-135, respectively) and the anti-EGFRGLYCOMAB (SEQ ID NOs: 102 and 103) were tested in the human head andneck carcinoma cell line FaDu, intralingually injected into SCID mice.This tumor model was shown by IHC on fresh frozen tissue to be positivefor FAP. FaDu cells were originally obtained from ATCC (American TypeCulture Collection) and after expansion deposited in the Glycartinternal cell bank. The tumor cell line was routinely cultured in DMEMcontaining 10% FCS (Gibco), at 37° C. in a water-saturated atmosphere at5% CO₂. In vitro passage 9 was used for intralingual injection, at aviability of 95.8%. Twenty μl cell suspension (2×10⁵ FaDu cells in AimVmedium (Gibco)) was injected intralingually. Female SCID mice (Taconics,Denmark), aged 8-9 weeks at the start of the experiment were maintainedunder specific-pathogen-free conditions with daily cycles of 12 hlight/12 h darkness according to committed guidelines (GV-Solas; Felasa;TierschG). The experimental study protocol was reviewed and approved bylocal government (P 2008016). After arrival, animals were maintained forone week to get accustomed to new environment and for observation.Continuous health monitoring was carried out on a regular basis. Micewere injected intralingually on study day 0 with 2×10⁵ FaDu cells,randomized and weighed. One week after the tumor cell injection, micewere injected i.v. with the 28H1 IgG-IL2 qm immunoconjugate, the DP47GSIgG-IL2 qm immunoconjugate, the anti-EGFR GLYCOMAB, the combination ofthe 28H1 IgG-IL2 qm immunoconjugate and the anti-EGFR GLYCOMAB, or thecombination of the DP47GS IgG-IL2 qm immunoconjugate and the anti-EGFRGLYCOMAB once weekly for four weeks. All mice were injected i.v. with200 μl of the appropriate solution. Doses are specified in Table 2. Themice in the vehicle group were injected with PBS and the treatmentgroups with the 28H1 IgG-IL2 qm immunoconjugate, the DP47GS IgG-IL2 qmimmunoconjugate, the anti-EGFR GLYCOMAB, the combination of the 28H1IgG-IL2 qm immunoconjugate and the anti-EGFR GLYCOMAB, or thecombination of the DP47GS IgG-IL2 qm immunoconjugate and the anti-EGFRGLYCOMAB. To obtain the proper amount of immunoconjugate per 200 thestock solutions were diluted with PBS when necessary. FIG. 1A shows thatonly the combination of the FAP-targeted 28H1 IgG-IL2 qm immunoconjugateand the anti-EGFR GLYCOMAB mediated superior efficacy in terms ofenhanced median survival compared to the 28H1 IgG-IL2 qm immunoconjugateor the anti-EGFR GLYCOMAB alone. In contrast thereto, the combination ofthe untargeted DP47GS IgG-IL2 qm and the anti-EGFR GLYCOMAB did not showsuperiority over the single agent administration (FIG. 1B).

TABLE 2 Concentration Compound Dose/mouse Formulation buffer (mg/mL)Anti-EGFR 625 μg  20 mM His/HisCl 26.65 GLYCOMAB 240 mM trehalose(=stock solution) 0.02% polysorbate 80  10 mM methionine pH 5.5 FAP 28H1 50 μg  20 mM histidine,  3.46 IgG-IL2 qm 140 mM NaCl, pH 6.0 (=stocksolution) untargeted  50 μg  20 mM histidine,  5.87 DP47GS 140 mM NaCl,pH 6.0 (=stock solution) IgG-IL2 qm

Example 2

In Vitro Boosting of NK Cell Killing Capacity by IL-2 Immunoconjugates

To determine the effect of immunoconjugates on NK cells, we assessed thekilling of tumor cells upon treatment with the immunoconjugates,particularly immunoconjugates comprising IL-2 as effector moiety. Forthis purpose, peripheral blood mononuclear cells (PBMCs) were isolatedaccording to standard procedures, using Histopaque-1077 (SigmaDiagnostics Inc., St. Louis, Mo., USA). In brief, venous blood was takenwith heparinized syringes from healthy volunteers. The blood was diluted2:1 with PBS not containing calcium or magnesium and layered onHistopaque-1077. The gradient was centrifuged at 450×g for 30 min atroom temperature (RT) without brake. The interphase containing the PBMCswas collected and washed with PBS in total three times (350×g followedby 300×g for 10 min at RT).

The isolated PBMCs were incubated with IL-2 (Proleukin) or IL-2immunoconjugates, added to the cell supernatant, for 45 h. Subsequently,the PBMCs were recovered and used for anti-EGFR GLYCOMAB-mediated ADCCof A549 cells at an E:T of 10:1, for 4 h. Target cell killing wasdetected by measuring LDH release into the cell supernatants (RocheCytotoxicity Detection Kit LDH). FIG. 2 shows the overall A549 tumorcell killing by PBMCs, pre-treated or not with 0.57 nM (A) or 5.7 nM (B)FAP-targeted 28H1 IgG-IL2 qm immunoconjugate or IL-2 (Proleukin), in thepresence of different concentrations of anti-EGFR GLYCOMAB. The graphsshow that immunoconjugate pre-treatment of effector cells results in agreater increase in target cell killing with increasing concentrationsof GLYCOMAB, as compared to untreated effector cells.

Example 3

LS174T Colorectal Xenograft Model

The CEA-targeted CH1A1A IgG-IL2 qm immunoconjugate (SEQ ID NOs:136-138), the anti-EGFR GLYCOMAB (SEQ ID NOs: 102 and 103) and cetuximabwere tested in the human colorectal LS174T cell line, intrasplenicallyinjected into SCID-human FcγRIII transgenic mice. This tumor model wasshown by IHC on fresh frozen tissue to be positive for CEA. LS174T cells(human colon carcinoma cells) were originally obtained from ECACC(European Collection of Cell Culture) and after expansion deposited inthe Glycart internal cell bank. LS174T were cultured in MEM Eagle'smedium containing 10% FCS (PAA Laboratories, Austria), 1% GLUTAMAX(L-alanyl-L-glutamine) and 1% MEM Non-Essential Amino Acids (Sigma). Thecells were cultured at 37° C. in a water-saturated atmosphere at 5% CO₂.In vitro passage 19 or 23 was used for intrasplenic injection, at aviability of 99%. A small incision was made at the left abdominal siteof anesthetized SCID FcγRIII transgenic mice. Thirty microliters cellsuspension (2×10⁶ LS174T cells in AimV medium) was injected through theabdominal wall just under the capsule of the spleen. Skin wounds wereclosed using clamps or resolvable sutures. Female SCID FcγRIIItransgenic mice; aged 8-9 weeks at the start of the experiment(purchased from Taconics, Denmark) were maintained underspecific-pathogen-free conditions with daily cycles of 12 h light/12 hdarkness according to committed guidelines (GV-Solas; Felasa; TierschG).The experimental study protocol was reviewed and approved by localgovernment (P 2008016, P2011/128). After arrival, animals weremaintained for one week to get accustomed to the new environment and forobservation. Continuous health monitoring was carried out on a regularbasis. Mice were injected intrasplenically on study day 0 with 2×10⁶LS174T cells, randomized and weighed. One week after the tumor cellinjection mice were injected i.v. with the CH1A1A IgG-IL2 qmimmunoconjugate, the anti-EGFR GLYCOMAB, cetuximab, the combination ofthe CH1A1A IgG-IL2 qm immunoconjugate and the anti-EGFR GLYCOMAB or thecombination of the CH1A1A IgG-IL2 qm immunoconjugate and cetuximab onceweekly for three weeks. All mice were injected i.v. with 200 μl of theappropriate solution. Doses are specified in Table 3. The mice in thevehicle group were injected with PBS and the treatment groups with theCH1A1A IgG-IL2 qm immunoconjugate or the anti-EGFR GLYCOMAB, cetuximab,the combination of the CH1A1A IgG-IL2 qm immunoconjugate and theanti-EGFR GLYCOMAB, or the combination of the CH1A1A IgG-IL2 qmimmunoconjugate and cetuximab. To obtain the proper amount ofimmunoconjugate per 200 the stock solutions were diluted with PBS whennecessary. FIG. 3 and Tables 3A and 3B show that the combination of theCH1A1A IgG-IL2 qm immunoconjugate and the anti-EGFR GLYCOMAB mediatedsuperior efficacy in terms of enhanced median and overall survivalcompared to the CH1A1A IgG-IL2 qm immunoconjugate alone, the anti-EGFRGLYCOMAB alone, cetuximab alone, or the combination of the CH1A1AIgG-IL2 qm immunoconjugate and cetuximab.

TABLE 3 Concentration Compound Dose/mouse Formulation buffer (mg/mL)Anti-EGFR 500 μg  20 mM His/HisCl 25.3 GLYCOMAB 240 mM trehalose (=stocksolution) 0.02% TWEEN (polysorbate) 20 pH 6.0 CH1A1A  40 μg  20 mMhistidine,  4.27 IgG-IL2 qm (FIG. 3A) or 140 mM NaCl, pH 6.0 (=stocksolution)  20 μg (FIG 3B) Cetuximab 500 μg  10 mM Na-citrate,  4.36NaCl, glycine, TWEEN (=stock solution) (polysorbate) 80, pH 5.5

TABLE 3A Summary of survival data corresponding to FIG. 3A. MedianOverall Treatment survival (days) survival Vehicle  24 0/8 CH1A1AIgG-IL2 qm  33 0/8 Anti-EGFR GLYCOMAB  40 0/8 CH1A1A IgG-IL2 qm + 1413/8 anti-EGFR GLYCOMAB

TABLE 3B Summary of survival data corresponding to FIG. 3B. MedianOverall Treatment survival (days) survival Vehicle 29 0/8 CH1A1A IgG-IL2qm 35 0/8 Cetuximab 39 0/8 CH1A1A IgG-IL2 qm + 53 2/8 Cetuximab(ongoing) (ongoing)

Example 4

A549 Lung Xenograft Model

The CEA-targeted CH1A1A IgG-IL2 qm immunoconjugate (SEQ ID NOs:136-138), the anti-EGFR GLYCOMAB (SEQ ID NOs: 102 and 103) and cetuximabwere tested in the human NSCLC cell line A549, injected i.v. intoSCID-human FcγRIII transgenic mice.

The A549 non-small cell lung carcinoma cells were originally obtainedfrom ATCC (CCL-185) and after expansion deposited in the Roche-Glycartinternal cell bank. The tumor cell line was routinely cultured in DMEMcontaining 10% FCS (Gibco) at 37° C. in a water-saturated atmosphere at5% CO₂. Passage 8 was used for transplantation, at a viability of97-98%. 5×10⁶ cells per animal were injected i.v. into the tail vein in200 μl of Aim V cell culture medium (Gibco). Female SCID-FcγRIII mice(Roche-Glycart; Switzerland), aged 8-9 weeks at the start of theexperiment (bred at Charles Rivers, Lyon, France) were maintained underspecific-pathogen-free condition with daily cycles of 12 h light/12 hdarkness according to committed guidelines (GV-Solas; Felasa; TierschG).The experimental study protocol was reviewed and approved by localgovernment (P 2011/128). After arrival, animals were maintained for oneweek to get accustomed to the new environment and for observation.Continuous health monitoring was carried out on a regular basis.

Mice were injected i.v. on study day 0 with 5×10⁶ A549 cells, randomizedand weighed. One week (FIG. 4A) or two weeks (FIG. 4B) after the tumorcell injection, mice were injected i.v. with the CH1A1A IgG-IL2 qmimmunoconjugate, the anti-EGFR GLYCOMAB, cetuximab, the combination ofthe CH1A1A IgG-IL2 qm immunoconjugate and the anti-EGFR GLYCOMAB, or thecombination of the CH1A1A IgG-IL2 qm immunoconjugate and cetuximab onceweekly for three weeks. All mice were injected i.v. with 200 μl of theappropriate solution. Doses are specified in Table 4. The mice in thevehicle group were injected with PBS and the treatment group with theCH1A1A IgG-IL2 qm immunoconjugate, the anti-EGFR GLYCOMAB, thecombination of the CH1A1A IgG-IL2 qm immunoconjugate and the anti-EGFRGLYCOMAB, or the combination of the CH1A1A IgG-IL2 qm immunoconjugateand cetuximab. To obtain the proper amount of immunoconjugate per 200the stock solutions were diluted with PBS when necessary. FIG. 4 andTables 4A and 4B show that the combination of the CH1A1A IgG-IL2 qmimmunoconjugate and the anti-EGFR GLYCOMAB mediates superior efficacy interms of enhanced median and overall survival compared to the respectiveimmunoconjugate, the anti-EGFR GLYCOMAB or cetuximab alone, as well asthe combination of the CH1A1A IgG-IL2 qm immunoconjugate and cetuximab.

TABLE 4 Concentration Compound Dose Formulation buffer (mg/mL) CH1A1A 20 μg  20 mM histidine,  4.27 IgG-IL2 qm 140 mM NaCl, (=stock solution)pH 6.0 Anti-EGFR 500 μg  20 mM His/HisCl, 25.3 GLYCOMAB 240 mMtrehalose, (=stock solution) 0.02% TWEEN (polysorbate) 20, pH 6.0Cetuximab 500 μg  10 mM Na-citrate,  4.36 NaCl, glycine, TWEEN (=stocksolution) (polysorbate) 80, pH 5.5

TABLE 4A Summary of survival data corresponding to FIG. 4A. MedianOverall Treatment survival (days) survival Vehicle  53 0/9 CH1A1AIgG-IL2 qm 103 0/9 Anti-EGFR GLYCOMAB 211 2/9 CH1A1A IgG-IL2 qm + notreached 9/9 anti-EGFR GLYCOMAB

TABLE 4B Summary of survival data corresponding to FIG. 4B. MedianOverall Treatment survival (days) survival Vehicle 49 0/10 CH1A1AIgG-IL2 qm 64 0/10 Cetuximab 68 0/10 CH1A1A IgG-IL2 qm + 91 0/10Cetuximab

Example 5

LS174T Colorectal Xenograft Model

The CEA-targeted CH1A1A IgG-IL2 qm immunoconjugate (SEQ ID NOs: 136-138)and the anti-Her3 GLYCOMAB (SEQ ID NOs: 142 and 146) were tested in thehuman colorectal LS174T cell line, intrasplenically injected intoSCID-human FcγRIII transgenic mice.

LS174T cells (human colon carcinoma cells) were originally obtained fromECACC (European Collection of Cell Culture) and after expansiondeposited in the Roche-Glycart internal cell bank. LS174T were culturedin MEM Eagle's medium containing 10% FCS (PAA Laboratories, Austria), 1%GLUTAMAX (L-alanyl-L-glutamine) and 1% MEM Non-Essential Amino Acids(Sigma). The cells were cultured at 37° C. in a water-saturatedatmosphere at 5% CO₂. In vitro passage 21 was used for intrasplenicinjection, at a viability of 97.9%. A small incision was made at theleft abdominal site of anesthetized SCID-human FcγRIII transgenic mice.Thirty microliters (2×10⁶ LS174T cells in AimV medium) cell suspensionwas injected through the abdominal wall just under the capsule of thespleen. Skin wounds were closed using resolvable sutures.

Female SCID-human FcγRIII transgenic mice; aged 8-9 weeks at the startof the experiment (Roche-Glycart, Switzerland) were maintained underspecific-pathogen-free conditions with daily cycles of 12 h light/12 hdarkness according to committed guidelines (GV-Solas; Felasa; TierschG).The experimental study protocol was reviewed and approved by localgovernment (P 2011/128). After arrival, animals were maintained for oneweek to get accustomed to the new environment and for observation.Continuous health monitoring was carried out on a regular basis.

Mice were injected intrasplenically on study day 0 with 2×10⁶ LS174Tcells, randomized and weighed. One week after the tumor cell injectionmice were injected i.v. with the CH1A1A IgG-IL2 qm immunoconjugate, theanti-Her3 GLYCOMAB or the combination of the CH1A1A IgG-IL2 qmimmunoconjugate and the anti-Her3 GLYCOMAB once weekly for three weeks.

All mice were injected i.v. with 200 μl of the appropriate solution.Doses are specified in Table 5. The mice in the vehicle group wereinjected with PBS and the treatment groups with the CH1A1A IgG-IL2 qmimmunoconjugate, the anti-Her3 GLYCOMAB or the combination of the CH1A1AIgG-IL2 qm immunoconjugate and the anti-Her3 GLYCOMAB. To obtain theproper amount of immunoconjugate per 200 μl, the stock solutions werediluted with PBS when necessary. FIG. 5 and Table 5A show that thecombination of the CH1A1A IgG-IL2 qm immunoconjugate and the anti-Her3GLYCOMAB mediated superior efficacy in terms of enhanced median survivalcompared to the CH1A1A IgG-IL2 qm immunoconjugate or the anti-Her3GLYCOMAB alone.

TABLE 5 Formulation Concentration Compound Dose buffer (mg/mL) Anti-Her3200 μg 10.0 GLYCOMAB (=stock solution) CH1A1A  20 μg  20 mM histidine,13.60 IgG-IL2 qm 140 mM NaCl, pH 6.0 (=stock solution)

TABLE 5A Summary of survival data corresponding to FIG. 5. MedianOverall Treatment survival (days) survival Vehicle 24 0/10 CH1A1AIgG-IL2 qm 25 0/10 Anti-Her3 GLYCOMAB 27 0/10 CH1A1A IgG-IL2 qm + 340/10 anti-Her3 GLYCOMAB

Example 6

ACHN Renal Carcinoma Xenograft Model

The FAP-targeted 28H1 IgG-IL2 immunoconjugate comprising the IL-2quadruple mutant (qm) (SEQ ID NOs: 125, 126 and 129) and the anti-EGFRGLYCOMAB (SEQ ID NOs: 102 and 103) were tested in the human renal cellline ACHN, intrarenally injected into SCID-human FcγRIII transgenicmice.

ACHN cells (human renal adenocarcinoma cells) were originally obtainedfrom ATCC (American Type Culture Collection) and after expansiondeposited in the Roche-Glycart internal cell bank. ACHN were cultured inDMEM containing 10% FCS. The cells were cultured at 37° C. in awater-saturated atmosphere at 5% CO₂. In vitro passage 22 was used forintrarenal injection, at a viability of 96.4%. A small incision (2 cm)was made at the right flank and peritoneal wall of anesthetizedSCID-human FcγRIII transgenic mice. Fifty μl (1×10⁶ ACHN cells in AimVmedium) cell suspension was injected 2 mm subcapsularly in the kidney.Skin wounds and peritoneal wall were closed using resolvable sutures.

Female SCID-human FcγRIII transgenic mice; aged 8-9 weeks at the startof the experiment (Roche-Glycart, Switzerland) were maintained underspecific-pathogen-free conditions with daily cycles of 12 h light/12 hdarkness according to committed guidelines (GV-Solas; Felasa; TierschG).The experimental study protocol was reviewed and approved by localgovernment (P 2011/128). After arrival, animals were maintained for oneweek to get accustomed to new environment and for observation.Continuous health monitoring was carried out on a regular basis.

Mice were injected intrarenally on study day 0 with 1×10⁶ ACHN cells,randomized and weighed. One week after the tumor cell injection, micewere injected i.v. with vehicle, anti-EGFR-GLYCOMAB, the combination ofthe 28H1 IgG-IL2 immunoconjugate and the anti-EGFR-GLYCOMAB, or thecombination of Proleukin® and the anti-EGFR-GLYCOMAB. The EGFR-GLYCOMABand the 28H1 IgG-IL2 immunoconjugate were dose once a week for 3 weeks.Proleukin® was injected daily from Monday to Friday for 3 weeks.

All mice were injected i.v. with 200 μl of the appropriate solution.Doses are specified in Table 6. The mice in the vehicle group wereinjected with PBS and the treatment groups with anti-EGFR-GLYCOMAB, thecombination of the 28H1 IgG-IL2 immunoconjugate and theanti-EGFR-GLYCOMAB, or the combination of Proleukin® and theanti-EGFR-GLYCOMAB. To obtain the proper amount of immunoconjugate per200 μl, the stock solutions were diluted with PBS when necessary.

FIG. 6 and Table 6A show that the combination of the 28H1 IgG-IL2immunoconjugate and the anti-EGFR-GLYCOMAB mediated superior efficacy interms of enhanced median and overall survival compared toanti-EGFR-GLYCOMAB alone and the combination of Proleukin® and theanti-EGFR-GLYCOMAB.

TABLE 6 Formulation Concentration Compound Dose buffer (mg/mL)Anti-EGFR-  625 μg  20 mM His/HisCl 26.65 GLYCOMAB 240 mM trehalose(=stock solution) 0.02% TWEEN (polysorbate) 20, pH 6.0 28H1 IgG-   78 μg 20 mM histidine,  3.46 IL2 140 mM NaCl, pH 6.0 (=stock solution)Proleukin ® 22.2 μg  1 (=stock solution)

TABLE 6A Summary of survival data corresponding to FIG. 6. MedianOverall Treatment survival (days) survival Vehicle  59 0/7 Anti-EGFRGLYCOMAB 155 0/7 Anti-EGFR GLYCOMAB + 174 0/7 Proleukin ® 28H1 IgG-IL2qm + anti- not reached 7/7 EGFR GLYCOMAB

Example 7

In Vitro Boosting of NK Cell Killing Capacity and NK Cell CD25 and CD69Expression by IL-2 Immunoconjugates

As in Example 2, we assessed the killing of tumor cells (LS174T) by NKcells upon treatment with an immunoconjugate, particularly animmunoconjugate comprising IL-2 as effector moiety, and a GLYCOMAB, inthis case an anti-Her3 GLYCOMAB. Target cell killing was detected bymeasuring LDH release into the cell supernatant.

PBMCs were isolated from fresh blood. Briefly, blood was diluted 3:1with PBS. About 30 ml of the blood/PBS mixture was stacked on 15 ml ofHistopaque (Sigma) and centrifuged for 30 min at 450 g for 30 minwithout brake. The lymphocytes were collected with a 5 ml pipette into50 ml tubes containing PBS. The tubes were filled up to 50 ml with PBSand centrifuged for 10 min at 350 g. The supernatant was discarded, thepellet re-suspended in 50 ml PBS and centrifuged for 10 min at 300 g.The washing step was repeated once. The cells were counted andre-suspended in pre-warmed RPMI containing 1% glutamine and 10% FCS with1×10⁶ cells per ml. The cells were incubated overnight at 37° C. On thenext day the cells were harvested and counted.

LS174T (ECACC #87060401) is a human Caucasian colon adenocarcinoma cellline. The cells were cultured in EMEM containing 1% glutamine, 1%non-essential amino acids and 10% FCS and splitted every two to threedays before reaching confluence. LS174T cells were detached usingtrypsin. The cells were counted and viability was checked. The viabilityof the cells before the assay was 99.4%. The cells were re-suspended intheir respective medium at 0.3×10⁶ per ml. 100 μl of the cell suspensionwas seeded into a 96 well cell culture treated flat bottom plate andincubated overnight at 37° C. On the next day the supernatant wasremoved from the cells. Then 50 μl of the diluted anti-Her3 GLYCOMAB(SEQ ID NOs: 142 and 146) at 1000 ng/ml, 100 ng/ml and 10 ng/ml ormedium was added to the respective wells. Then, 50 μl of theCEA-targeted CH1A1A IgG-IL2 qm immunoconjugate (SEQ ID NOs: 136-138) atthe indicated concentrations (see FIG. 7) or medium was added per well.After 10 minutes incubation at room temperature 100 n1 of PBMCs at 3×10⁶cells per ml or medium were added to reach a final volume of 200 μl perwell. The cells were incubated for 24 hours in the incubator. After theincubation the plate was centrifuged for 4 minutes at 400 g and thesupernatant was collected. 50 μl per well of the supernatant were usedto measure LDH release (Roche Cytotoxicity Detection Kit LDH). Theremaining supernatant was stored at −20° C. until further use. The cellswere re-suspended in FACS buffer and stored at 4° C. before startingwith the FACS staining (see below)

FIG. 7 shows the overall LS174T cell killing by PBMCs upon treatmentwith anti-Her3 GLYCOMAB alone (left panel), the CH1A1A IgG-IL-2 qmimmunoconjugate alone (right panel) or the combination of the CH1A1AIgG-IL-2 qm immunoconjugate with the anti-Her3 GLYCOMAB (right panel).

The PBMCs were harvested after 24 h and used for FACS analysis of NKcell CD25 and CD69 expression. The cells were centrifuged for 4 min at400 g and washed once with PBS containing 0.1% BSA (FACS buffer). Then20 μl per well of the antibody mix was added to the cells. The cellswere incubated for 30 min in the fridge. Afterwards the cells werewashed twice with FACS buffer and re-suspended in 200 μl FACS buffercontaining 2% PFA per well. The analysis was performed using the BD FACSFortessa, and the following antibody mix: CD3 PE/Cy7 (BioLegend,#300420; diluted 1:40), CD56 APC (BioLegend #318310; diluted 1:20), CD69Brilliant Violet 421 (BioLegend #310929; diluted 1:40), CD25 PE (BDBioscience #557138; diluted 1:20).

FIG. 8 shows expression of CD25 (A) or CD69 (B) on NK cells upontreatment with anti-Her3 GLYCOMAB alone (left panel), the CH1A1AIgG-IL-2 qm immunoconjugate alone (right panel) or the combination ofthe CH1A1A IgG-IL-2 qm immunoconjugate with the anti-Her3 GLYCOMAB(right panel).

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, the descriptions and examples should not be construed aslimiting the scope of the invention. The disclosures of all patent andscientific literature cited herein are expressly incorporated in theirentirety by reference.

The invention claimed is:
 1. A method of treating a carcinoembryonicantigen (CEA)-expressing or Fibroblast Activation Protein (FAP):expressing cancer in an individual comprising administering to theindividual a combination of: (a) an immunoconjugate comprising: (i) afirst full-length IgG antibody which specifically binds to a CEA antigenor a FAP antigen, wherein the first full-length IgG antibody comprises ahuman IgG Fc region, and is engineered to have reduced effector functioncompared to a corresponding non-engineered antibody and comprises in theheavy chain one or more amino acid substitutions selected from the groupconsisting of S228P, E233P, L234A, L235A, L235E, N297A, N297D, P331S,and P329G according to EU numbering, and (ii) a mutant interleukin-2(IL-2) effector moiety, wherein the mutant IL-2 effector moiety is humanIL-2 comprising the amino acid substitutions F42A, Y45A, and L72Gaccording to the amino acid residue positions of SEQ ID NO:1, and (b) asecond full-length IgG antibody which specifically binds to EpidermalGrowth Factor Receptor (EGFR), wherein the second full-length IgGantibody is cetuximab, in a therapeutically effective amount.
 2. Themethod of claim 1, wherein the first full-length IgG antibody is an IgG₁antibody.
 3. The method of claim 1, wherein the mutant IL-2 effectormoiety shares an amino- or carboxy-terminal peptide bond with the firstfull-length IgG antibody.
 4. The method of claim 1, wherein the firstfull-length IgG antibody is engineered to have reduced binding to anactivating Fc receptor, or reduced binding to human FcγRIIIa.
 5. Themethod of claim 1, wherein the first full-length IgG antibody comprisesan amino acid substitution at position P329 in the immunoglobulin heavychains according to EU numbering.
 6. The method of claim 1, wherein thefirst full-length IgG antibody comprises the amino acid substitutionsL234A, L235A and P329G in the immunoglobulin heavy chains according toEU numbering.
 7. The method of claim 1, wherein the immunoconjugateconsists essentially of the mutant IL-2 effector moiety and the firstfull-length IgG antibody, wherein the mutant IL-2 effector moiety isfused at its amino-terminal amino acid to the carboxy-terminus of one ofthe heavy chains of the first full-length IgG antibody, optionallythrough a peptide linker.
 8. The method of claim 1, wherein the effectorfunction is selected from the group of binding to an activating Fcreceptor, antibody-dependent cell-mediated cytotoxicity (ADCC),antibody-dependent cellular phagocytosis (ADCP), complement-dependentcytotoxicity (CDC), and cytokine secretion.
 9. The method of claim 1,wherein the effector function is binding to an activating Fc receptorand/or ADCC.
 10. The method of claim 1, wherein the individual is ahuman.
 11. The method of claim 1, wherein the first full-length IgGantibody comprises the heavy chain variable region sequence of SEQ IDNO: 114 and the light chain variable region sequence of SEQ ID NO: 115.12. The method of claim 1, wherein the first full-length IgG antibodycomprises: (a) the heavy chain variable region sequence of SEQ ID NO: 12and the light chain variable region sequence of SEQ ID NO: 11; (b) theheavy chain variable region sequence of SEQ ID NO: 17 and the lightchain variable region sequence of SEQ ID NO: 16; (c) the heavy chainvariable region sequence of SEQ ID NO: 47 and the light chain variableregion sequence of SEQ ID NO: 46; (d) the heavy chain variable regionsequence of SEQ ID NO: 63 and the light chain variable region sequenceof SEQ ID NO: 62; or (e) the heavy chain variable region sequence of SEQID NO: 67 and the light chain variable region sequence of SEQ ID NO: 66.13. The method of claim 1, wherein the mutant IL-2 additionallycomprises an amino acid mutation at a position corresponding to position3 according to the amino acid residue positions of SEQ ID NO:1, whicheliminates the 0-glycosylation site of IL-2.
 14. The method of claim 13,wherein the additional amino acid mutation is an amino acid substitutionreplacing a threonine residue with an alanine residue.
 15. The method ofclaim 1, wherein the mutant IL-2 comprises the sequence of SEQ ID NO: 2.16. The method of claim 1, wherein the cancer is selected from the groupconsisting of lung cancer, colorectal cancer, renal cancer, and head andneck cancer.
 17. The method of claim 1, wherein the first full-lengthIgG antibody binds to CEA and comprises: a) in the heavy chain variabledomain a CDR1 of SEQ ID NO: 108, a CDR2 of SEQ ID NO: 109, and a CDR3 ofSEQ ID NO: 110, and b) in the light chain variable domain a CDR1 of SEQID NO: 111, a CDR2 of SEQ ID NO: 112, and a CDR3 of SEQ ID NO:
 113. 18.The method of claim 1, wherein the human IgG Fc region of the firstfull-length IgG antibody is a human IgG1 Fc region.
 19. A method ofstimulating effector cell function in an individual having acarcinoembryonic antigen (CEA)-expressing or Fibroblast ActivationProtein (FAP)-expressing cancer, comprising administering to theindividual a combination of (a) an immunoconjugate comprising: (i) afirst full-length IgG antibody which specifically binds to a CEA antigenor a FAP antigen, wherein the first full-length IgG antibody comprises ahuman IgG Fc region, and is engineered to have reduced effector functioncompared to a corresponding non-engineered antibody and comprises in theheavy chain one or more amino acid substitutions selected from the groupconsisting of S228P, E233P, L234A, L235A, L235E, N297A, N297D, P331S,and P329G according to EU numbering, and (ii) a mutant interleukin-2(IL-2) effector moiety, wherein the mutant IL-2 effector is human IL-2comprising the amino acid substitutions F42A, Y45A, and L72G accordingto the amino acid residue positions of SEQ ID NO:1, and (b) a secondfull-length IgG antibody which specifically binds to Epidermal GrowthFactor Receptor (EGFR), wherein the second full-length IgG antibody iscetuximab, in an amount effective to stimulate effector cell function.20. The method of claim 19, wherein the first full-length IgG antibodybinds to CEA and comprises: a) in the heavy chain variable domain a CDR1of SEQ ID NO: 108, a CDR2 of SEQ ID NO: 109, and a CDR3 of SEQ ID NO:110, and b) in the light chain variable domain a CDR1 of SEQ ID NO: 111,a CDR2 of SEQ ID NO: 112, and a CDR3 of SEQ ID NO:
 113. 21. The methodof claim 19, wherein the first full-length IgG antibody comprises theheavy chain variable region sequence of SEQ ID NO: 114 and the lightchain variable region sequence of SEQ ID NO:
 115. 22. The method ofclaim 19, wherein the mutant IL-2 comprises the sequence of SEQ ID NO:2.